THE ROLE OF THE PHOSPHOINOSITIDE PATHWAY IN HORMONAL REGULATION OF THE EPITHELIAL SODIUM CHANNEL

In summary, insulin and aldosterone stimulate phosphatidylinositol phosphorylation, thus indicating the existence of a regulated protein at or before the PI3-kinase step. Aldosterone induces the synthesis of sgk, a downstream element of the PI pathway. Sgk is necessary, but not rate-limiting, for aldosterone- and insulin-stimulated Na+ transport. However, the enzyme appears to be rate-limiting for the natriferic action of ADH. Insulin-stimulated Na+ transport, an acute response, is dependent on PI3-kinase activity but the magnitude of the response is not altered by a cellular excess of sgk. ADH-stimulated transport is not dependent on PI3-kinase but is potentiated by an excess of sgk. The foregoing data indicate that the PI pathway is involved in several steps of the natriferic action of hormones and intersects with other pathways which regulate ENaC. Furthermore, the data are consistent with the hypothesis that activation of PI3-kinase may ultimately stimulate channel insertion as well as regulate channel endocytosis. Both of these phenomena can result in an increase of ENaC-mediated Na+ transport

Phosphoinositide lipid second messengers: new paradigms for transepithelial signal transduction

Multiple forms of phosphatidylinositol are generated by differential phosphorylation of the inositol headgroup. These phosphoinositides, specifically PI(4,5)P2, have been implicated as modulators in a variety of transport processes. The data indicate that phosphoinositides can modulate transporters directly or via the activation of down-stream signaling components. The phosphoinositide pathway has been linked to changes in transporter kinetics, intracellular signaling, membrane targeting and membrane stability. Recent results obtained for several of the well-characterized transport systems suggest the need to reassess the role of PI(4,5)P2 and question whether lower abundance forms of the phosphoinositides, notably PI(3,4,5)P3 (PIP3) and PI(3,4)P2, are the pertinent transport regulators. In contrast to PI(4,5)P2, these latter forms represent a dynamic, regulated pool, the characteristics of which are more compatible with the nature of signaling intermediates. A recently described, novel transepithelial signaling pathway has been demonstrated for PIP3 in which a signal initiated on the basolateral membrane is transduced to the apical membrane entirely within the planar face of the inner leaflet of the plasma membrane. The new paradigms emerging from recent studies may be widely applicable to transporter regulation in other cell types and are particularly relevant for signaling in polarized cells

Hormonal regulation of the epithelial Na+ channel: From amphibians to mammals

High-resistance epithelia derived from amphibian sources such as frog skin, toad urinary bladder, and the A6 Xenopus laevis kidney cell line have been widely used to elucidate the underlying mechanisms involved in the regulation of vectorial ion transport. More recently, the isolation of high-resistance mammalian cell lines has provided model systems in which to study differences and similarities between the regulation of ion transporter function in amphibian and mammalian renal epithelia. In the present study, we have compared the natriferic (Na+ retaining) responses to aldosterone, insulin, and vasotocin/vasopressin in the A6 and mpkCCDcl4 (mouse principal cells of the kidney cortical collecting duct) cell lines. The functional responses of the epithelial Na+ channel (ENaC) to hormonal stimulation were remarkably similar in both the amphibian and mammalian lines. In addition, insulin- and aldosterone-stimulated, reabsorptive Na+ transport in both cell lines requires the presence of functional PI3-kinase

EGF stimulates IClswell by a redistribution of proteins involved in cell volume regulation.

Background: ICln is a multifunctional protein involved in the generation of chloride currents activated during regulatory volume decrease (RVD) after cell swelling (IClswell). Growth factor receptors play a key role in different cellular processes and epidermal growth factor (EGF) regulates swelling-activated chloride permeability. Aim: We set out to investigate if the EGF-induced upregulation of IClswell could be explained by a rearrangement of ICln subcellular distribution and interaction with its molecular partners. Methods: NIH-3T3 fibroblasts were serum-deprived for 24 hours and stimulated with EGF (40 ng/ml) for 30 minutes. IClswell activation, ICln distribution and interaction with its molecular partner HSPC038 were assessed by whole cell patch clamp and fluorescence resonance energy transfer (FRET). Results: EGF treatment significantly enhanced the direct molecular interaction between ICln and HSPC038 and also resulted in an increase of ICln and HSPC038 association with the plasma membrane. Importantly, these events are associated with a significant increase of IClswell. Conclusions: The present data indicate that EGF might exert its role in the modulation of volume-sensitive chloride currents in part through activation and translocation of ICln and HSPC038 to the plasma membrane

Vasopressin Regulates the Phosphorylation State of AMP-activated Protein Kinase (AMPK) in MDCK-C7 Cells

AMP-activated protein kinase (AMPK) is a regulatory kinase coupling cellular metabolism with ion transport. Madin-Darby Canine Kidney-Clone 7 (MDCK-C7) cells possess characteristics of the renal principal cell type, express the cystic fibrosis transmembrane regulator and the epithelial Na(+) channel, and display NPPB and amiloride-sensitive transepithelial transport when stimulated with [Arg(8)]-vasopressin. [Arg(8)]-vasopressin binding to its receptor on the basolateral membrane of MDCK-C7 results in cAMP production, activation of cAMP-dependent protein kinase A (PKA), and increases in Cl(-) and Na(+) transport. Ussing-style electrophysiology showed that the PKA inhibitor, H89, blocked Cl(-) and Na(+) transport. Unexpectedly, [Arg(8)]-vasopressin stimulation resulted in the dephosphorylation of pAMPK(thr172). H89 did not prevent this, suggesting that the dephosphorylation is independent of PKA. 24 hour, but not 15 minute, incubation with the AMPK activator, AICAR, also blocked [Arg(8)]-vasopressin-stimulated currents. Contrary to previous studies, immunoblotting revealed that AICAR did not increase abundance of the active, phosphorylated form of AMPK (pAMPK(thr172)); although, AICAR treatment significantly blocked [Arg(8)]-vasopressin -stimulated cAMP production. [Arg(8)]-vasopressin still caused pAMPK(thr172) dephosphorylation in the presence of AICAR, suggesting that this effect is also independent of cAMP. In summary, these data suggest [Arg(8)]-vasopressin regulates AMPK phosphorylation and that AICAR inhibits ion transport independently of AMPK in MDCK-C7 cells

PPARγ agonists do not directly enhance basal or insulin-stimulated Na+ transport via the epithelial Na+ channel

Selective agonists of peroxisome proliferator-activated receptor gamma (PPARgamma) are anti-diabetic drugs that enhance cellular responsiveness to insulin. However, in some patients, fluid retention, plasma volume expansion, and edema have been observed. It is well established that insulin regulates Na(+) reabsorption via the epithelial sodium channel (ENaC) located in the distal tubule. Therefore, we hypothesized that these agonists may positively modulate insulin-stimulated ENaC activity leading to increased Na(+) reabsorption and fluid retention. Using electrophysiological techniques, dose-response curves for insulin-mediated Na(+) transport in the A6, M-1, and mpkCCD(cl4) cell lines were performed. Each line demonstrated hormone efficacy within physiological concentration ranges and, therefore, can be used to monitor clinically relevant effects of pharmacological agents which may affect electrolyte transport. Immunodetection and quantitative PCR analyses showed that each cell line expresses viable and functional PPARgamma receptors. Despite this finding, two PPARgamma agonists, pioglitazone and GW7845 did not directly enhance basal or insulin-stimulated Na(+) flux via ENaC, as shown by electrophysiological methodologies. These studies provide important results, which eliminate insulin-mediated ENaC activation as a candidate mechanism underlying the fluid retention observed with PPARgamma agonist use

A FRET-Based Approach for Quantitative Evaluation of Forskolin-Induced Pendrin Trafficking at the Plasma Membrane in Bronchial NCI H292 Cells

Background: Human pendrin (SLC26A4, PDS) is an integral membrane protein acting as an electroneutral anion exchanger. Loss of function mutations in pendrin protein cause Pendred syndrome, a disorder characterized by sensorineural deafness and a partial iodide organification defect that may lead to thyroid goiter. Additionally, pendrin up-regulation could play a role in the pathogenesis of several diseases including bronchial asthma and chronic obstructive pulmonary disease (COPD). Therefore, monitoring the plasma membrane abundance and trafficking of pendrin in the context of a living cell is crucially important. Methods: Trafficking of pendrin to the plasma membrane was monitored by fluorescence resonance energy transfer (FRET), a physical phenomenon occurring between two fluorophores (the FRET donor and acceptor) located in close spatial proximity. Because the efficiency of the energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, FRET is extremely sensitive to small changes in distance between the donor and acceptor and is therefore a powerful tool to determine protein-protein interactions. Results: FRET studies revealed that forskolin-induced cAMP production is associated with a significant increase of pendrin expression at plasma membrane, which is paralleled by a decrease in intracellular pH. Pendrin transposition to the membrane is accompanied with a partial depolymerization of actin cytoskeleton via Rho-GTPase inhibition. Conclusion: Trafficking to the plasma membrane is critical in the regulation of pendrin activity. Therefore, reliable tools for monitoring and quantifying this phenomenon are highly desirable

Case report: metoclopramide induced acute dystonic reaction in adolescent CYP2D6 poor metabolizers

Metoclopramide is indicated for the management of gastroesophageal reflux, gastric stasis, nausea, and vomiting. Metoclopramide-induced acute dystonic reactions (MIADRs), along with repetitive involuntary protrusion of the tongue, are well-known phenomena in children and young adults that may appear after the first dose. The drug is primarily metabolized via oxidation by the cytochrome P450 enzyme CYP2D6 and to a lesser extent by CYP3A4 and CYP1A2. A recommendation to decrease metoclopramide dosing in patients with severely limited to no CYP2D6 activity (i.e., poor metabolizers, PMs) is included in the drug label. It is important to note, however, that a requirement or recommendation for pre-emptive testing for CYP2D6 metabolizer status is not included in the drug label. We present two cases of acute dystonia in two non-consanguineous male adolescents: one following metoclopramide and cimetidine administration in a 14-year-old to treat gastroesophageal reflux, and another following metoclopramide and pantoprazole administration in a 17-year-old with acute gastroenteritis. A retrospective pharmacogenetic analysis revealed both patients as CYP2D6 PMs

60kDa Lysophospholipase, a New Sgk1 Molecular Partner Involved in the Regulation of ENaC

The serum- and glucocorticoid-regulated kinase (Sgk1) is essential for hormonal regulation of ENaC-mediated sodium transport and is involved in the transduction of growth-factor-dependent cell survival and proliferation. The identification of molecular partners for Sgk1 is crucial for the understanding of its mechanisms of action. We performed a yeast two-hybrid screening based on a human kidney cDNA library to identify molecular partners of Sgk1. As a result the screening revealed a specific interaction between Sgk1 and a 60 kDa Lysophospholipase (LysoLP). LysoLP is a poorly characterized enzyme that, based on sequence analysis, might possess lysophospholipase and asparaginase activities. We demonstrate that LysoLP has indeed a lysophospholipase activity and affects metabolic functions related to cell proliferation and regulation of membrane channels. Moreover we demonstrate in the Xenopus oocyte expression system that LysoLP downregulates basal and Sgk1-dependent ENaC activity. In conclusion LysoLP may represent a new player in the regulation of ENaC and Sgk1-dependent signaling

4th ESPT Conference:pharmacogenomics and personalized medicine - research progress and clinical implementation

The Fourth European Society of Pharmacogenomics and Personalized Therapy biennial conference was organized in collaboration with the Italian Society of Personalized Medicine (SIMeP) and was held at Benedictine Monastery of San Nicolò l'Arena in Catania, Sicily (Italy) on 4-7 October 2017. The congress addressed the research progress and clinical implementation in pharmacogenomics and personalized medicine. The Fourth European Society of Pharmacogenomics and Personalized Therapy congress brought together leading international scientists and healthcare professionals actively working in the fields of pharmacogenomics and personalized therapy. Altogether, 25 speakers in 15 session comprehensively covered broad spectrum of pharmacogenetics and pharmacogenomics research, clinical applications in different clinical disciplines attended by 270 delegates