Extracellular urea concentration modulates cAMP production in the mouse MTAL

International audienceIonic reabsorption along the ascending limb of Henle's loop (TAL) is controlled by hormonal stimulation. Most of the hormones that affect this reabsorption regulate ionic transporter activity via cAMP, and some of these hormonal actions have been shown to be modulated by interstitial osmolarity. We studied the early effects of increasing extracellular urea concentration on the production of cAMP induced by arginine vasopressin (AVP) and forskolin in a suspension of medullary portions of TAL (MTAL) prepared from mouse kidney. The addition of urea, performed fifteen minutes before adenylyl cyclase stimulation, decreased both AVP- and forskolin-induced cAMP production. This effect, observed both in the presence and the absence of phosphodiesterase inhibition, was optimal with 300 mmol/liter urea. Addition of urea to the extracellular medium disturbed several cellular parameters, but the decrease in cAMP production appeared to be mediated by the activation of both the protein kinase A and a phosphatase rather than by the modifications in phospholipid metabolism. Since cAMP is the major cytosolic transductional factor in MTAL cells, urea present in the medullary interstitium may thus be considered as an important modulator of hormonal actions in this segment of the nephron

Inhibition of the Calcium Release-activated Calcium (CRAC) Current in Jurkat T Cells by the HIV-1 Envelope Protein gp160

International audienceThe HIV-1 envelope glycoprotein gp120/160 has pleiotropic effects on T cell function. We investigated whether Ca(2+) signaling, a crucial step for T cell activation, was altered by prolonged exposure of Jurkat T cells to gp160. Microfluorometric measurements showed that Jurkat cells incubated with gp160 had smaller (approximately 40%) increases in [Ca(2+)](i) in response to phytohemagglutinin and had a reduced Ca(2+) influx (approximately 25%). gp160 had similar effects on Jurkat cells challenged with thapsigargin. We used the patch clamp technique to record the Ca(2+) current, which is responsible for Ca(2+) influx and has properties of the calcium release-activated Ca(2+) current (I(CRAC)). gp160 reduced I(CRAC) by approximately 40%. The inhibitory effects of gp160 were antagonized by staurosporine (0.1 microm), an inhibitor of protein-tyrosine kinases and protein kinase Cs (PKCs), and by Gö 6976 (5 microm), an inhibitor acting especially on PKC alpha and PKC beta I. 12-O-Tetradecanoyl phorbol 13-acetate (16 nm), a PKC activator, reproduced the effects of gp160 in untreated cells. A Western blotting analysis of PKC isoforms alpha, beta I, delta, and zeta showed that only the cellular distribution of PKC alpha and -beta I were significantly modified by gp160. In addition, gp160 was able to modify the subcellular distribution of PKC alpha and PKC beta I caused by phytohemagglutinin. Therefore the reduction in I(CRAC) caused by prolonged incubation with gp160 is probably mediated by PKC alpha or -beta I

An unexpected effect of TNF-α on F508del-CFTR maturation and function

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An unexpected effect of TNF-α on F508del-CFTR maturation and function [v1; ref status: indexed, http://f1000r.es/5jf]

Cystic fibrosis (CF) is a multifactorial disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR), which encodes a cAMP-dependent Cl- channel. The most frequent mutation, F508del, leads to the synthesis of a prematurely degraded, otherwise partially functional protein. CFTR is expressed in many epithelia, with major consequences in the airways of patients with CF, characterized by both fluid transport abnormalities and persistent inflammatory responses. The relationship between the acute phase of inflammation and the expression of wild type (WT) CFTR or F508del-CFTR is poorly understood. The aim of the present study was to investigate this effect. The results show that 10 min exposure to TNF-alpha (0.5-50ng/ml) of F508del-CFTR-transfected HeLa cells and human bronchial cells expressing F508del-CFTR in primary culture (HBE) leads to the maturation of F508del-CFTR and induces CFTR chloride currents. The enhanced CFTR expression and function upon TNFα is sustained, in HBE cells, for at least 24 h. The underlying mechanism of action involves a protein kinase C (PKC) signaling pathway, and occurs through insertion of vesicles containing F508del-CFTR to the plasma membrane, with TNFα behaving as a corrector molecule. In conclusion, a novel and unexpected action of TNFα has been discovered and points to the importance of systematic studies on the roles of inflammatory mediators in the maturation of abnormally folded proteins in general and in the context of CF in particular

Factors influencing readthrough therapy for frequent cystic fibrosis premature termination codons

Premature termination codons (PTCs) are generally associated with severe forms of genetic diseases. Readthrough of in-frame PTCs using small molecules is a promising therapeutic approach. Nonetheless, the outcome of preclinical studies has been low and variable. Treatment efficacy depends on: 1) the level of drug-induced readthrough, 2) the amount of target transcripts, and 3) the activity of the recoded protein. The aim of the present study was to identify, in the cystic fibrosis transmembrane conductance regulator (CFTR) model, recoded channels from readthrough therapy that may be enhanced using CFTR modulators. First, drug-induced readthrough of 15 PTCs was measured using a dual reporter system under basal conditions and in response to gentamicin and negamycin. Secondly, exon skipping associated with these PTCs was evaluated with a minigene system. Finally, incorporated amino acids were identified by mass spectrometry and the function of the predicted recoded CFTR channels corresponding to these 15 PTCs was measured. Nonfunctional channels were subjected to CFTR-directed ivacaftor-lumacaftor treatments. The results demonstrated that CFTR modulators increased activity of recoded channels, which could also be confirmed in cells derived from a patient. In conclusion, this work will provide a framework to adapt treatments to the patient's genotype by identifying the most efficient molecule for each PTC and the recoded channels needing co-therapies to rescue channel function

Characterization of two rat models of cystic fibrosis-KO and F508del CFTR-Generated by Crispr-Cas9

Genetically engineered animals are essential for gaining a proper understanding of the disease mechanisms of cystic fibrosis (CF). The rat is a relevant laboratory model for CF because of its zootechnical capacity, size, and airway characteristics, including the presence of submucosal glands