Anoctamin 1 (Ano1) is required for glucose-induced membrane potential oscillations and insulin secretion by murine ß-cells.

Anions such as Cl(-) and HCO3 (-) are well known to play an important role in glucose-stimulated insulin secretion (GSIS). In this study, we demonstrate that glucose-induced Cl(-) efflux from ß-cells is mediated by the Ca(2+)-activated Cl(-) channel anoctamin 1 (Ano1). Ano1 expression in rat ß-cells is demonstrated by reverse transcriptase-polymerase chain reaction, western blotting, and immunohistochemistry. Typical Ano1 currents are observed in whole-cell and inside-out patches in the presence of intracellular Ca(++): at 1 µM, the Cl(-) current is outwardly rectifying, and at 2 µM, it becomes almost linear. The relative permeabilities of monovalent anions are NO3 (-) (1.83 ± 0.10) > Br(-) (1.42 ± 0.07) > Cl(-) (1.0). A linear single-channel current-voltage relationship shows a conductance of 8.37 pS. These currents are nearly abolished by blocking Ano1 antibodies or by the inhibitors 2-(5-ethyl-4-hydroxy-6-methylpyrimidin-2-ylthio)-N-(4-(4-methoxyphenyl)thiazol-2-yl)acetamide (T-AO1) and tannic acid (TA). These inhibitors induce a strong decrease of 16.7-mM glucose-stimulated action potential rate (at least 87 % on dispersed cells) and a partial membrane repolarization with T-AO1. They abolish or strongly inhibit the GSIS increment at 8.3 mM and at 16.7 mM glucose. Blocking Ano1 antibodies also abolish the 16.7-mM GSIS increment. Combined treatment with bumetanide and acetazolamide in low Cl(-) and HCO3 (-) media provokes a 65 % reduction in action potential (AP) amplitude and a 15-mV AP peak repolarization. Although the mechanism triggering Ano1 opening remains to be established, the present data demonstrate that Ano1 is required to sustain glucose-stimulated membrane potential oscillations and insulin secretion.

Short forms of Ste20-related proline/alanine-rich kinase (SPAK) in the kidney are created by aspartyl aminopeptidase (Dnpep)-mediated proteolytic cleavage.

The Ste20-related kinase SPAK regulates sodium, potassium, and chloride transport in a variety of tissues. Recently, SPAK fragments, which lack the catalytic domain and are inhibitory to Na(+) transporters, have been detected in kidney. It has been hypothesized that the fragments originate from alternative translation start sites, but their precise origin is unknown. Here, we demonstrate that kidney lysate possesses proteolytic cleavage activity toward SPAK. Ion exchange and size exclusion chromatography combined with mass spectrometry identified the protease as aspartyl aminopeptidase. The presence of the protease was verified in the active fractions, and recombinant aspartyl aminopeptidase recapitulated the cleavage pattern observed with kidney lysate. Identification of the sites of cleavage by mass spectrometry allowed us to test the function of the smaller fragments and demonstrate their inhibitory action toward the Na(+)-K(+)-2Cl(-) cotransporter, NKCC2.

A novel Ste20-related proline/alanine-rich kinase (SPAK)-independent pathway involving calcium-binding protein 39 (Cab39) and serine threonine kinase with no lysine member 4 (WNK4) in the activation of Na-K-Cl cotransporters.

Na(+)-dependent chloride cotransporters (NKCC1, NKCC2, and NCC) are activated by phosphorylation to play critical roles in diverse physiological responses, including renal salt balance, hearing, epithelial fluid secretion, and volume regulation. Serine threonine kinase WNK4 (With No K = lysine member 4) and members of the Ste20 kinase family, namely SPAK and OSR1 (Ste20-related proline/alanine-rich kinase, Oxidative stress-responsive kinase) govern phosphorylation. According to present understanding, WNK4 phosphorylates key residues within SPAK/OSR1 leading to kinase activation, allowing SPAK/OSR1 to bind to and phosphorylate NKCC1, NKCC2, and NCC. Recently, the calcium-binding protein 39 (Cab39) has emerged as a binding partner and enhancer of SPAK/OSR1 activity, facilitating kinase autoactivation and promoting phosphorylation of the cotransporters. In the present study, we provide evidence showing that Cab39 differentially interacts with WNK4 and SPAK/OSR1 to switch the classic two kinase cascade into a signal kinase transduction mechanism. We found that WNK4 in association with Cab39 activates NKCC1 in a SPAK/OSR1-independent manner. We discovered that WNK4 possesses a domain that bears close resemblance to the SPAK/OSR1 C-terminal CCT/PF2 domain, which is required for physical interaction between the Ste20 kinases and the Na(+)-driven chloride cotransporters. Modeling, yeast two-hybrid, and functional data reveal that this PF2-like domain located downstream of the catalytic domain in WNK4 promotes the direct interaction between the kinase and NKCC1. We conclude that in addition to SPAK and OSR1, WNK4 is able to anchor itself to the N-terminal domain of NKCC1 and to promote cotransporter activation.

Physiology and pathophysiology of SLC12A1/2 transporters.

The electroneutral Na(+)-K(+)-Cl(-) cotransporters NKCC1 (encoded by the SLC12A2 gene) and NKCC2 (SLC12A1 gene) belong to the Na(+)-dependent subgroup of solute carrier 12 (SLC12) family of transporters. They mediate the electroneutral movement of Na(+) and K(+), tightly coupled to the movement of Cl(-) across cell membranes. As they use the energy of the ion gradients generated by the Na(+)/K(+)-ATPase to transport Na(+), K(+), and Cl(-) from the outside to the inside of a cell, they are considered secondary active transport mechanisms. NKCC-mediated transport occurs in a 1Na(+), 1K(+), and 2Cl(-) ratio, although NKCC1 has been shown to sometimes mediate partial reactions. Both transporters are blocked by bumetanide and furosemide, drugs which are commonly used in clinical medicine. NKCC2 is the molecular target of loop diuretics as it is expressed on the apical membrane of thick ascending limb of Henle epithelial cells, where it mediates NaCl reabsorption. NKCC1, in contrast, is found on the basolateral membrane of Cl(-) secretory epithelial cells, as well as in a variety of non-epithelial cells, where it mediates cell volume regulation and participates in Cl(-) homeostasis. Following their molecular identification two decades ago, much has been learned about their biophysical properties, their mode of operation, their regulation by kinases and phosphatases, and their physiological relevance. However, despite this tremendous amount of new information, there are still so many gaps in our knowledge. This review summarizes information that constitutes consensus in the field, but it also discusses current points of controversy and highlights many unanswered questions.

A primary culture of distal convoluted tubules expressing functional thiazide-sensitive NaCl transport.

Studying the molecular regulation of the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) is important for understanding how the kidney contributes to blood pressure regulation. Until now, a native mammalian cell model to investigate this transporter remained unknown. Our aim here is to establish, for the first time, a primary distal convoluted tubule (DCT) cell culture exhibiting transcellular thiazide-sensitive Na(+) transport. Because parvalbumin (PV) is primarily expressed in the DCT, where it colocalizes with NCC, kidneys from mice expressing enhanced green-fluorescent protein (eGFP) under the PV gene promoter (PV-eGFP-mice) were employed. The Complex Object Parametric Analyzer and Sorter (COPAS) was used to sort fluorescent PV-positive tubules from these kidneys, which were then seeded onto permeable supports. After 6 days, DCT cell monolayers developed transepithelial resistance values of 630 ± 33 Ω·cm(2). The monolayers also established opposing transcellular concentration gradients of Na(+) and K(+). Radioactive (22)Na(+) flux experiments showed a net apical-to-basolateral thiazide-sensitive Na(+) transport across the monolayers. Both hypotonic low-chloride medium and 1 µM angiotensin II increased this (22)Na(+) transport significantly by four times, which could be totally blocked by 100 µM hydrochlorothiazide. Angiotensin II-stimulated (22)Na(+) transport was also inhibited by 1 µM losartan. Furthermore, NCC present in the DCT monolayers was detected by immunoblot and immunocytochemistry studies. In conclusion, a murine primary DCT culture was established which expresses functional thiazide-sensitive Na(+)-Cl(-) transport.

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.

Inhibition of insulin-stimulated hydrogen peroxide production prevents stimulation of sodium transport in A6 cell monolayers.

Insulin-stimulated sodium transport across A6 cell (derived from amphibian distal nephron) monolayers involves the activation of a phosphatidylinositol (PI) 3-kinase. We previously demonstrated that exogenous addition of H2O2 to the incubation medium of A6 cell monolayers provokes an increase in PI 3-kinase activity and a subsequent rise in sodium transport (Markadieu N, Crutzen R, Blero D, Erneux C, Beauwens R. Am J Physiol Renal Physiol 288: F1201-F1212, 2005). We therefore questioned whether insulin would produce an intracellular burst of H2O2 leading to PI 3-kinase activation and subsequent increase in sodium transport. An acute production of reactive oxygen species (ROS) in A6 cells incubated with the oxidation-sensitive fluorescent probe 5,6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate was already detected after 2 min of insulin stimulation. This fluorescent signal and the increase in sodium transport were completely inhibited in monolayers incubated with peggylated catalase, indicating that H2O2 is the main intracellular ROS produced upon insulin stimulation. Similarly, preincubation of monolayers with different chelators of either superoxide (O2(*-); nitro blue tetrazolium, 100 microM) or H2O2 (50 microM ebselen), or blockers of NADPH oxidase (Nox) enzymes (diphenyleneiodonium, 5 microM; phenylarsine oxide, 1 microM and plumbagin, 30 microM) prevented both insulin-stimulated H2O2 production and insulin-stimulated sodium transport. Furthermore, diphenyleneiodonium pretreatment inhibited the recruitment of the p85 PI 3-kinase regulatory subunit in an anti-phosphotyrosine immunoprecipitate in insulin-stimulated cells. In contrast, PI-103, an inhibitor of class IA PI 3-kinase, inhibited insulin-stimulated sodium transport but did not significantly reduce insulin-stimulated H2O2 production. Taken together, our data suggest that insulin induces an acute burst of H2O2production which participates in an increase in phosphatidylinositol 3,4,5-trisphosphate production and subsequently stimulation of sodium transport.

Selection of in-phase or out-of-phase synchronization in a model based on global coupling of cells undergoing metabolic oscillations.

On the basis of experimental observations, it has been suggested that glycolytic oscillations underlie the pulsatile secretion of insulin by pancreatic beta cells, with a periodicity of about 13 min. If beta cells within an islet are synchronized through gap junctions, the question arises as to how beta cells located in different islets of Langerhans synchronize to produce oscillations in plasma levels of insulin. We address this question by means of a minimal model that incorporates the secretion of insulin by cells undergoing glycolytic oscillations. Global coupling and synchronization result from the inhibition exerted by insulin on the production of glucose, which serves as the substrate for metabolic oscillations. Glycolytic oscillations are described by a simple two-variable model centered on the product-activated reaction catalyzed by the allosteric enzyme phosphofructokinase. We obtain bifurcation diagrams for the cases in which insulin secretion is controlled solely by the product or by the substrate of the metabolic oscillator. Remarkably, we find that the oscillating cells in these conditions synchronize, respectively, in phase or out of phase. Numerical simulations show that in-phase and out-of-phase synchronization can sometimes coexist when insulin release is controlled by both the substrate and the product of the metabolic oscillator. The results provide an example of a system in which the selection of in-phase or out-of-phase synchronization is governed by the nature of the coupling between the intracellular oscillations and the secretion of the biochemical signal through which the oscillating cells are globally coupled.

Synthesis and biological activity of phosphatidylinositol-3,4,5-trisphosphorothioate.

Metabolically-stabilized analogs of PtdIns(3,4,5)P(3) have shown long-lived agonist activity for cellular events mediated by this phosphoinositide. We describe an efficient method for the total asymmetric synthesis of the trisphosphorothioate (PT) analog of PtdIns(3,4,5)P(3). Intracellular delivery of dipalmitoyl PtdIns(3,4,5)PT(3)-mimicked insulin in activating 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.