Long-term CFTR inhibition modulates 15d-prostaglandin J2 in human pulmonary cells.

Prostaglandins, the products of arachidonic acid release and oxidation by phospholipase A(2) and cyclooxygenases (COX) 1 and 2 respectively, are known as important inflammation mediators. However, their diversity in structure, properties and cell specificity make their physiological function difficult to define. In the lung, the prostaglandin D(2) (PGD(2)) metabolite 15d-PGJ(2) is known to modulate the properties of a large number of intracellular compounds, leading to both pro- and anti-inflammatory effects.

Oxidative stress induces unfolding protein response and inflammation in nasal polyposis.

Background: Nasal polyposis, a chronic inflammatory disease affecting the upper airways, is a valuable and accessible model to investigate the mechanisms underlying chronic inflammation. The main objective of this study was to investigate a potential involvement of the unfolded protein response (UPR) in the context of oxidative stress and inflammation in nasal epithelial cells from nasal polyps (NP).

Methods: Epithelial cells from NP (n = 20) and normal mucosa (Controls, n = 15) in primary culture were analyzed by global proteomic approach and cell biology techniques for the glucose-regulated protein 78 (GRP78), the spliced X-box-binding protein 1 (sXBP-1), the glucose-regulated protein 94 (GRP94), and the calreticulin (immunoblot, mass spectrometry, immunocytochemistry).

GSH monoethyl ester rescues mitochondrial defects in cystic fibrosis models.

Cystic fibrosis (CF), a multisystem disease caused by CFTR (cystic fibrosis transmembrane conductance regulator) gene mutations, is associated with an abnormal inflammatory response and compromised redox homeostasis in the airways. Recent evidence suggests that dysfunctional CFTR leads to redox imbalance and to mitochondrial reduced glutathione (mtGSH) depletion in CF models. This study was designed to investigate the consequences of mtGSH depletion on mitochondrial function and inflammatory response.

Eicosanoid release is increased by membrane destabilization and CFTR inhibition in Calu-3 cells.

The antiinflammatory protein annexin-1 (ANXA1) and the adaptor S100A10 (p11), inhibit cytosolic phospholipase A2 (cPLA2alpha) by direct interaction. Since the latter is responsible for the cleavage of arachidonic acid at membrane phospholipids, all three proteins modulate eicosanoid production. We have previously shown the association of ANXA1 expression with that of CFTR, the multifactorial protein mutated in cystic fibrosis.

Control of basal CFTR gene expression by bicarbonate-sensitive adenylyl cyclase in human pulmonary cells.

The CFTR protein, encoded by the gene whose mutations induce Cystic Fibrosis, is an anion channel devoted mainly to chloride and bicarbonate transmembrane transport, but which also regulates transport of several other ions. Moreover, it is implicated in the cell response to inflammation, and, reciprocally, cftr gene expression is modulated by inflammatory stimuli and transduction pathways. Looking for a control of CFTR expression by ionic conditions, we investigated the effect of altered extracellular bicarbonate ion concentration on CFTR expression in human pulmonary Calu-3 cells.

Membrane cholesterol content modulates ClC-2 gating and sensitivity to oxidative stress.

ClC-2 is a broadly expressed member of the voltage-gated ClC chloride channel family. In this study, we aimed to evaluate the role of the membrane lipid environment in ClC-2 function, and in particular the effect of cholesterol and ClC-2 distribution in membrane microdomains. Detergent-resistant and detergent-soluble microdomains (DSM) were isolated from stably transfected HEK293 cells by a discontinuous OptiPrep gradient.

Rescue of DeltaF508-CFTR (cystic fibrosis transmembrane conductance regulator) by curcumin: involvement of the keratin 18 network.

The most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, DeltaF508, causes retention of DeltaF508-CFTR in the endoplasmic reticulum and leads to the absence of CFTR Cl(-) channels in the plasma membrane. DeltaF508-CFTR retains some Cl(-) channel activity so increased expression of DeltaF508-CFTR in the plasma membrane can restore Cl(-) secretion deficiency. Recently, curcumin was shown to rescue DeltaF508-CFTR localization and function.

Cell-specific posttranscriptional regulation of CFTR gene expression via influence of MAPK cascades on 3'UTR part of transcripts.

Expression of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene, which contains the mutations responsible for CF, is regulated by cytokines (TNF-alpha and IL-1beta) in a cell-specific manner. TNF-alpha decreases CFTR mRNA in human colon cell lines (HT-29), but not in pulmonary cell lines (Calu-3), and IL-1beta increases it only in Calu-3 cells. We looked for the cytokine-induced posttranscriptional regulation of CFTR gene expression and studied the modulation of CFTR mRNA stability linked to its 3' untranslated sequence (3'UTR) in HT-29 and Calu-3 cells.

Effect of ouabain on CFTR gene expression in human Calu-3 cells.

We have previously shown that ouabain, which changes the electrochemical properties of cell membranes by inhibiting Na(+),K(+)-ATPase, induces the expression of multidrug resistance (MDR-1) gene in several human cell lines. Because the expressions of the MDR-1 and CFTR (which encodes the cAMP-activated Cl(-) channel associated with cystic fibrosis) genes are physiologically regulated in opposing directions, we wanted to determine whether ouabain also decreases CFTR transcripts and subsequently to analyze its mechanism of action. We found that the submicromolar concentrations of ouabain that increase MDR-1 mRNAs decrease the CFTR transcripts with analogous time-dependency in human pulmonary Calu-3 cells.

Drug resistance induced by ouabain via the stimulation of MDR1 gene expression in human carcinomatous pulmonary cells.

The inhibition of the Na+/K+-ATPase by cardiotonic drugs like ouabain deeply perturbs both the properties of the cell membrane and the ionic composition of the cytoplasm and hence alters fundamental cell reactions. These three types of reactions may be involved in the stimulation of multidrug resistance 1 (MDR-1) gene expression and the synthesis of permeability glycoprotein [P-glycoprotein (P-gp)]. We have determined whether ouabain, which binds to an extracellular motif of the Na+/K+-ATPase, stimulates MDR-1 gene expression by measuring both mRNA and protein and whether the resulting P-gp extrudes hydrophobic compounds and causes resistance to antimitotic agents.

NF-kappa B mediates up-regulation of CFTR gene expression in Calu-3 cells by interleukin-1beta.

Inflammation of the airways is a major feature of the inherited disease cystic fibrosis. Previous studies have shown that the pro-inflammatory cytokines tumor necrosis factor alpha and interferon gamma reduce the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene (CFTR) in HT-29 and T84 cells by acting post-transcriptionally. We have investigated the effect of the pro-inflammatory peptide interleukin 1beta (IL-1beta) on the expression of the CFTR in Calu-3 cells.

Aspirin and some other nonsteroidal anti-inflammatory drugs inhibit cystic fibrosis transmembrane conductance regulator protein gene expression in T-84 cells.

Cystic fibrosis (CF) is caused by mutations in the CF gene, which encodes CF transmembrane conductance regulator protein (CFTR), a transmembrane protein that acts as a cAMP-regulated chloride channel The disease is characterized by inflammation but the relationship between inflammation, abnormal transepithelial ion transport, and the clinical manifestations of CF are uncertain. The present study was undertaken to determine whether three nonsteroidal anti-inflammatory drugs (NSAIDs) (aspirin, ibuprofen, and indomethacin) modulate CFTR gene expression in T-84 cells. Treatment with NSAIDs reduced CFTR transcripts, and decreased cAMP-stimulated anion fluxes, an index of CFTR function.

Modulation of CFTR gene expression in HT-29 cells by extracellular hyperosmolarity.

Hypertonicity has pleiotropic effects on cell function, including activation of transporters and regulation of gene expression. It is important to investigate the action of hypertonicity on cystic fibrosis gene expression because cystic fibrosis transmembrane conductance regulator (CFTR), the cAMP-regulated Cl(-) channel, regulates ion transport across the secretory epithelia, which are often in a hypertonic environment. We found that adding >150 mosmol/l NaCl, urea, or mannitol to the culture medium reduced the amount of CFTR mRNA in colon-derived HT-29 cells in a time-dependent manner.

Functional evidence for a Ca2+/polyvalent cation sensor in the mouse thick ascending limb.

The effects of extracellular polyvalent cations on the cytosolic free Ca2+ concentration ([Ca2+]i) of isolated segments of the mouse nephron were investigated using fura 2 microfluorometry. Extracellular Ca2+ concentration ([Ca2+]o), gadolinium (Gd3+), and neomycin (Neo) increased the [Ca2+]i in cortical thick ascending limb (CTAL) tubules with effective doses (ED50) of approximately 3.5 mM for Ca2+, 20 microM for Gd3+, and 40 microM for Neo.

Extracellular urea concentration modulates cAMP production in the mouse MTAL.

Ionic 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.

Hypertonic NaCl enhances adenosine release and hormonal cAMP production in mouse thick ascending limb.

Adenosine 3',5'-cyclic monophosphate (cAMP), accumulated in the presence of adenosine, was measured in medullary portions of mouse thick ascending limbs of Henle's loop, suspended either in classic extracellular buffer or in the presence of added NaCl. Under control conditions (140 mmol/l NaCl), adenosine (< 10(-5) mol/l) and N6-cyclohexyladenosine, an A1 adenosine receptor agonist, inhibit the cAMP accumulation induced by arginine vasopressin (AVP). On the other hand, high concentrations of adenosine and CGS-21680, an A2 adenosine receptor agonist, stimulate cAMP formation.

Extracellular ATP and UTP trigger calcium entry in mouse cortical thick ascending limbs.

The effects of extracellular nucleotides on the cytosolic free Ca2+ concentration ([Ca2+]i) of mouse cortical thick ascending limb (CTAL) segments were investigated using the Ca(2+)-sensitive fluorescent probe fura 2. ATP (50% effective dose, ED50, 40 microM) transiently increased [Ca2+]i, while adenosine (a P1 purinoceptor agonist), N6-cyclohexyladenosine (an A1 agonist), AMP, ADP (a P2t agonist), beta, gamma-methyleneadenosine 5'-triphosphate (a P2x agonist), or 2-methylthioadenosine 5'-triphosphate (a P2y agonist) all had little or no effect. CTAL tubules were also sensitive to UTP.

Actions of isoproterenol in frog proximal tubules.

In the present work, we compared biochemical and electrophysiological actions of isoproterenol on frog proximal tubular cells by using tubule suspensions and giant entities obtained by cell fusion. Isoproterenol (ISO) dose-dependently stimulated cAMP production in tubule suspension and depolarized the "giant cell" membrane. Both effects were triggered by beta receptor occupancy, but strongly differed in their concentration-dependency, since depolarization occurred with an ISO concentration as low as 10(-12) mol/l whereas cAMP accumulation could be seen only with more than 10(-8) mol/l ISO.

[Arginine]vasopressin hydrolyses phosphoinositides in the medullary thick ascending limb of mouse nephron.

NaCl reabsorption across the thick ascending limb of Henle's loop (TAL) is stimulated by several hormones, in particular vasopressin acting through V2 receptors and cyclic AMP production. This study used suspensions of medullary TAL (mTAL) tubules from the mouse nephron to investigate the possibility that, besides activating adenylyl cyclase, vasopressin also stimulates phospholipase C via V1 receptor occupancy. Two different methods, phosphoinositide labelling and inositol trisphosphate (InsP3) radioimmunoassay, were used to show that [arginine]vasopressin (AVP) rapidly stimulated the formation of InsP3, which peaked at 200%-250% of control within the first minute of incubation with 10 nmol/l vasopressin at 37 degrees C, and declined to basal level after 5-10 min.

Hyperactivation of phospholipase C does not support the enhanced proliferation of aortic smooth muscle cells from spontaneously hypertensive rats.

When cultured in the presence of fetal calf serum, arterial smooth muscle cells from spontaneously hypertensive rats (SHR) proliferate more rapidly and are more numerous at confluency than cells from normotensive Wistar-Kyoto (WKY) animals. The phenomenon has been demonstrated in several laboratories but its molecular origin remains unclear. On the other hand phospholipase C activation and c-fos transcription are early events able to trigger cell mitosis.

[Role of external calcium on the growth of aortic smooth muscle cells in SHR].

Cultured aortic smooth muscle cells from SHR proliferate more actively than cells normotensive control animals. This experimental data may be related to the hypertensive arteriopathy which mainly proceeds from media dystrophy made of hypertrophy, hyperplasia and excessive protein secretion of the smooth muscle cells. In order to precise the molecular cause of the phenomenon and the eventual action of calcium channel blockers on the development of this organic characteristic of hypertension, we have compared the responses of cultured cells from both SH and WKY rats to various agents in the absence or presence of verapamil.

Angiotensin II-induced proliferation of aortic myocytes in spontaneously hypertensive rats.

In order to determine whether the morphological modifications observed in arterial media of spontaneously hypertensive rats (SHR) could be induced by an abnormal response of the smooth muscle cells to vasoactive agents, we studied the action of angiotensin (Ang) II on cultured aortic smooth muscle cells from both SHR and Wistar-Kyoto rats (WKY). Under our experimental conditions, Ang II exerts a mitogenic action on SHR cells, whereas its effect is very weak on WKY cells. Phospholipase C activation and c-fos and c-myc proto-oncogene expressions induced by Ang II are considerably enhanced in SHR cells, and these abnormalities may be linked to an increased number of Ang II receptors.

Enhanced cell proliferation in essential hypertension.

In order to define the molecular mechanism involved in enhancement of spontaneously hypertensive rat (SHR) cell proliferation, we have compared the actions of fetal calf serum (FCS) and angiotensin II on both SHR and Wistar-Kyoto (WKY) rat aortic smooth muscle cells. Both compounds are more mitogenic in SHR cells than in controls. However, phospholipase C (PLC) hyperresponsiveness can be seen only under angiotensin stimulation, as are the expressions of c-jun, c-fos, and c-myc.

Hypertension and atherosclerosis.

Membrane phospholipase C (PLC) activation is induced by the interaction of numerous vasoactive hormones and growth factors with their receptors. Two products are liberated: inositol triphosphate (IP3) and diacyglycerol (DG). The first product liberates intracellular calcium from its stores in the sarcoplasmic reticulum and the second one activates a phosphokinase, which triggers a transmembrane Na+/H+ exchange.

Role of nuclear proto-oncogenes in the proliferation of aortic smooth muscle cells in spontaneously hypertensive rats.

In order to define the molecular mechanism involved in the enhancement of spontaneously hypertensive rat (SHR) cell proliferation, we compared the actions of fetal calf serum and angiotensin II on both SHR and Wistar-Kyoto rat (WKY) aortic smooth muscle cells. Both compounds were more mitogenic on the SHR cells than on the controls. However, phospholipase C hyper-responsiveness was present only after angiotensin stimulation.