[The impact of hypoxia on the ion channels in cystic fibrosis bronchial epithelial cells]. / Impact de l'hypoxie sur les canaux ioniques dans des cellules épithéliales bronchiques mucoviscidosiques.

Cystic fibrosis is a disease caused by a mutation on the CFTR gene coding for a chloride channel. The dominant mutation F508del eliminates the CFTR protein at the surface of epithelial cells, causing an accumulation of viscous mucus in the airways. In advanced stages of the disease, respiratory failure is associated with cellular hypoxia. Our project aims not only to describe the impact of hypoxia on ion channels and to highlight the underlying signaling pathways involved, but also to test the effectiveness of current CF treatments under the above-mentioned conditions.

Mechanistic analysis of nonoxygenated hypothermic machine perfusion's protection on warm ischemic kidney uncovers greater eNOS phosphorylation and vasodilation.

Protection of endothelial cell function may explain the benefits of nonoxygenated hypothermic machine perfusion (MP) for marginal kidney preservation. However, this hypothesis remains to be tested with a preclinical model. We postulated that MP protects the nitric oxide (NO) signaling pathway, altered by static cold storage (CS), and improves renal circulation recovery compared to CS. The endothelium releases the vasodilator NO in response to flow via either increased endothelial NO synthase (eNOS) expression (KLF2-dependent) or activation of eNOS by phosphorylation (via Akt, PKA or AMPK). Using a porcine model of kidney transplantation, including 1 h of warm ischemia and preserved 24 h by CS or MP (n=5), we reported that MP did not alter the cortical levels of KLF2 and eNOS at the end of preservation, but significantly increased eNOS activating phosphorylation compared to CS. eNOS phosphorylation appeared AMPK-dependent and was concomitant to an increased NO-dependent vasodilation of renal arteries measured, ex situ, at the end of preservation. In vivo, laser Doppler showed that cortical microcirculation was improved at reperfusion in MP kidneys. In conclusion, we demonstrate for the first time, in a large-animal model, that MP protects the NO signaling pathway, confirming the value of MP for marginal kidney preservation.

Roscovitine is a proteostasis regulator that corrects the trafficking defect of F508del-CFTR by a CDK-independent mechanism.

BACKGROUND AND PURPOSE: The most common mutation in cystic fibrosis (CF), F508del, causes defects in trafficking, channel gating and endocytosis of the CF transmembrane conductance regulator (CFTR) protein. Because CF is an orphan disease, therapeutic strategies aimed at improving mutant CFTR functions are needed to target the root cause of CF. EXPERIMENTAL APPROACH: Human CF airway epithelial cells were treated with roscovitine 100 µM for 2 h before CFTR maturation, expression and activity were examined. The mechanism of action of roscovitine was explored by recording the effect of depleting endoplasmic reticulum (ER) Ca(2+) on the F508del-CFTR/calnexin interaction and by measuring proteasome activity. KEY RESULTS: Of the cyclin-dependent kinase (CDK) inhibitors investigated, roscovitine was found to restore the cell surface expression and defective channel function of F508del-CFTR in human CF airway epithelial cells. Neither olomoucine nor (S)-CR8, two very efficient CDK inhibitors, corrected F508del-CFTR trafficking demonstrating that the correcting effect of roscovitine was independent of CDK inhibition. Competition studies with inhibitors of the ER quality control (ERQC) indicated that roscovitine acts on the calnexin pathway and on the degradation machinery. Roscovitine was shown (i) to partially inhibit the interaction between F508del-CFTR and calnexin by depleting ER Ca(2+) and (ii) to directly inhibit the proteasome activity in a Ca(2+) -independent manner. CONCLUSIONS AND IMPLICATIONS: Roscovitine is able to correct the defective function of F508del-CFTR by preventing the ability of the ERQC to interact with and degrade F508del-CFTR via two synergistic but CDK-independent mechanisms. Roscovitine has potential as a pharmacological therapy for CF.

Evaluation of the Bronchorelaxant, Genotoxic, and Antigenotoxic Effects of Cassia alata L.

Aqueous-ethanolic extract of Cassia alata (AECal) and its derived fractions obtained through liquid-liquid fractionation were evaluated for their bronchorelaxant, genotoxic, and antigenotoxic effects. Contractile activity of rats' tracheas in the presence of tested materials, as well as its modifications with different inhibitors and blockers, was isometrically recorded. The antigenotoxic potential of AECal was evaluated on cyclophosphamide- (CP-) induced genotoxicity in the rat. Animals were pretreated with the extract, then liver comet assay was performed. AECal and its chloroformic fractions (CF-AECal) relaxed the contraction induced by Ach, but both were significantly less potent in inhibiting contraction induced by KCl (30 mM; 80 mM). Propranolol, indomethacin, L-NAME, methylene blue, and glibenclamide did not modify the relaxant effect of CF-AECal. TEA altered the response of trachea to CF-AECal. CF-AECal caused a rightward shift without affecting the E max in cumulative concentration-response curves of Ach only at low concentrations. In animals pretreated with the extract, the percentage of CP-induced DNA damage decreased. Our results suggest that (1) muscarinic receptors contribute at least in part to the relaxant effects of CF-AECal; (2) CF-AECal interferes with membrane polarization; and (3) AECal is not genotoxic in vivo and contains chemopreventive phytoconstituents offering protection against CP-induced genotoxicity.

From the vasodilator and hypotensive effects of an extract fraction from Agelanthus dodoneifolius (DC) Danser (Loranthaceae) to the active compound dodoneine.

AIM OF THE STUDY: Effects of the different fractions obtained by partition of ethanolic extract (EE) of Agelanthus dodoneifolius through column chromatography were investigated on rat blood pressure and aortic relaxation and compared to those observed in the presence of crude EE. MATERIALS AND METHODS: The acute hypotensive activity of EE, fractions and dodoneine, administrated intravenously, was evaluated in anaesthetized rats using the invasive method of blood pressure recording. Bioassay-guided fractionation using rat aorta pre-contracted by norepinephrine to monitor the relaxant activity led to the isolation of dodoneine. RESULTS: In normotensive rats, injection of EE (0.01-10 mg/kg) produced a dose-dependent decrease in both systolic and diastolic blood pressure without any significant change in heart rate. In a similar way, the EE (0.001-3 mg/mL) caused relaxation of rat pre-contracted aorta in a concentration-dependent manner. Fractionation of the EE afforded 14 fractions, F1-F14, that were tested on rat precontracted aortic rings. At the concentration level of 1 mg/mL, a maximum relaxation effect was observed for fractions F2-F5. F4 was the most effective to elicit a concentration-dependent relaxation effect with an ED(50)=160±1.1 µg/mL (n=5) and to decreased systolic and diastolic control pressure by 56.9% and 81.6% respectively. F4 contains most of the dihydropyranone dodoneine, with 93% of the sample mass. Dodoneine separated from this fraction was also able to decrease both systolic and diastolic arterial pressure by 32.5% and 38.7% at 100 µg/kg, respectively. CONCLUSION: For the first time, this study demonstrates the hypotensive property of the dodoneine present in Agelanthus dodoneifolius.

Identification of a novel water-soluble activator of wild-type and F508del CFTR: GPact-11a.

One of the major therapeutic strategy in cystic fibrosis aims at developing modulators of cystic fibrosis transmembrane conductance regulator (CFTR) channels. We recently discovered methylglyoxal alpha-aminoazaheterocycle adducts, as a new family of CFTR inhibitors. In a structure-activity relationship study, we have now identified GPact-11a, a compound able not to inhibit but to activate CFTR. Here, we present the effect of GPact-11a on CFTR activity using in vitro (iodide efflux, fluorescence imaging and patch-clamp recordings), ex vivo (short-circuit current measurements) and in vivo (salivary secretion) experiments. We report that GPact-11a: 1) is an activator of CFTR in several airway epithelial cell lines; 2) activates rescued F508del-CFTR in nasal, tracheal, bronchial, pancreatic cell lines and in human CF ciliated epithelial cells, freshly dissociated from lung samples; 3) stimulates ex vivo the colonic chloride secretion and increases in vivo the salivary secretion in cftr(+/+) but not cftr(-/-) mice; and 4) is selective for CFTR because its effect is inhibited by CFTR(inh)-172, GlyH-101, glibenclamide and GPinh-5a. To conclude, this work identifies a selective activator of wild-type and rescued F508del-CFTR. This nontoxic and water-soluble agent represents a good candidate, alone or in combination with a F508del-CFTR corrector, for the development of a CFTR modulator in cystic fibrosis.

Calcium sparks in muscle cells: interactive procedures for automatic detection and measurements on line-scan confocal images series.

In muscle cells, force development is controlled by Ca2+ ions, which are rapidly released from the sarcoplasmic reticulum (SR) during sarcolemmal depolarization. In addition to this synchronized spatially homogeneous calcium signal, localized quantal Ca2+ release events (sparks) have been recorded using laser scanning confocal fluorescence microscopy. Previously, algorithms without user intervention have been developed to automatically detect and analyse sparks on confocal line-scan (space-time: 512 x 512 pixels) single images. Here we present a computer program that we called "HARVest of Elementary Events" (HARVELE) developed to both analyse events on series of confocal images and follow sparks morphology from one site during several seconds. HARVELE, coded in the image-processing language IDL 5.3., can be applied on series of n images (512 x 512 x n) obtained from the same scanning line. Computing simultaneously entire series of images allows to measure, for each release site, the frequency and the morphology of release with the conventional amplitude, size, time and duration parameters defined for sparks analysis. The use of these procedures rapidly provides much information about the properties of calcium release sites in muscle cells population and can be applied on any elementary calcium events whatever the type of cell.

Prevention of pathology in mdx mice by expression of utrophin: analysis using an inducible transgenic expression system.

Duchenne muscular dystrophy results from the absence of dystrophin, a cytoskeletal protein. Previously, we have shown in a transgenic mouse model of the disease (mdx) that high levels of expression of the dystrophin-related protein, utrophin can prevent pathology. We developed a new transgenic mouse model where muscle specific utrophin expression was conditioned by addition of tetracycline in water. Transgene expression was turned on at different time points: in utero, at birth, 10 and 30 days after birth. We obtained moderate levels of expression, variable from fibre to fibre (mosaicism) but sufficient to induce a correct localization of the dystro-sarcoglycan complex. Histology revealed a reduction of necrotic foci and of the percentage of centronucleated fibres, which remained still largely above the normal level. Isometric force was not improved but the resistance to eccentric contractions was significantly stronger. When utrophin expression was activated 30 days after birth, improvements were marginal, suggesting that the age at which utrophin therapy is initiated could be an important factor. Our results also provide an unexpected insight into the pathogenesis of the dystrophinopathies. We observed a complete normalization of the characteristics of the mechano-sensitive/voltage-independent Ca(2+) channels (occurrence, open probabilities and Ca(2+) currents), while the classical markers of dystrophy were still abnormal. These observations question the role of increased Ca(2+) channel activity in initiating the dystrophic process. The new model shows that utrophin therapy, initiated after birth, can be effective, but the extent of correction of the various symptoms of dystrophinopathy critically depends on the amount of utrophin expressed.

Long-term study of Ca(2+) homeostasis and of survival in collagenase-isolated muscle fibres from normal and mdx mice.

Skeletal muscles of the mdx mouse lack dystrophin offering the possibility to study the role of intracellular Ca(2+) ions in fibre degeneration. Flexor digitorum brevis muscles of 3-month-old mdx and normal mice were dissociated with collagenase; fibres were maintained in culture for 6 days (d0 to d5) and their survival was assessed. Cytosolic [Ca(2+)], passive Mn(2+) influx (indicative of Ca(2+) influx) and activity of mechanosensitive/voltage-independent Ca(2+) channels were studied over the same period. Survival of normal fibres declined steadily from d0 to d3, but an acceleration of fibre death occurred in mdx fibres from d1 to d2. This could be greatly reduced but not abolished by lowering external [Ca(2+)] 10-fold. In the d0-d5 period, both mdx and normal fibres showed transient increases of Mn(2+) influx and activity of the Ca(2+) channels; these peaked at d1 and disappeared by d3-d4. Increases were always significantly larger in mdx fibres. Altogether, over the 6 days, 130 paired measurements of [Ca(2+)](i) and Mn(2+) influx were made on 68 fibres from mdx and 62 fibres from normal mice. In 90 % of the fibres, [Ca(2+)](i) remained within the 25-85 nM limits while Mn(2+) influx varied more than 10-fold. The median for Mn(2+) influx was 45 % greater in fibres from mdx mice than in fibres from control C57 mice. However, there was no significant difference between [Ca(2+)](i) medians in fibres from normal and mdx mice. Addition of 25-75 nM of a Ca(2+) ionophore (4-bromo-A23187) to the medium did not affect the level of cytosolic [Ca(2+)] in both types of fibres, while markedly increasing the rate of Mn(2+) influx, as expected. Thus, Ca(2+) homeostasis was equally robust in mdx and normal fibres. The remaining 10 % of the fibres showed, at d1, high levels of Mn(2+) influx and/or elevated [Ca(2+)](i) above 100 nM. This did not affect survival of normal fibres but was probably responsible of the increased death rate in mdx fibres.

Calcium currents and transients in co-cultured contracting normal and Duchenne muscular dystrophy human myotubes.

1. The goal of the present study was to investigate differences in calcium movements between normal and Duchenne muscular dystrophy (DMD) human contracting myotubes co-cultured with explants of rat spinal cord with attached dorsal root ganglia. Membrane potential, variations of intracellular calcium concentration and T- and L-type calcium currents were recorded. Further, a descriptive and quantitative study by electron microscopy of the ultrastructure of the co-cultures was carried out. 2. The resting membrane potential was slightly less negative in DMD (-61.4 +/- 1.1 mV) than in normal myotubes (-65.5 +/- 0.9 mV). Both types of myotube displayed spontaneous action potentials (mean firing frequency, 0.42 and 0.16 Hz, respectively), which triggered spontaneous calcium transients measured with Indo-1. 3. The time integral under the spontaneous Ca(2+) transients was significantly greater in DMD myotubes (97 +/- 8 nM s) than in normal myotubes (67 +/- 13 nM s). 4. The L- and T-type current densities estimated from patch-clamp recordings were smaller in DMD cells (2.0 +/- 0.5 and 0.90 +/- 0.19 pA pF(-1), respectively) than in normal cells (3.9 +/- 0.7 and 1.39 +/- 0.30 pA pF(-1), respectively). 5. The voltage-dependent inactivation relationships revealed a shift in the conditioning potential at which inactivation is half-maximal (V(h,0.5)) of the T- and L-type currents towards less negative potentials, from -72.1 +/- 0.7 and -53.7 +/- 1.5 mV in normal cells to -61.9 +/- 1.4 and -29.2 +/- 1.4 mV in DMD cells, respectively. 6. Both descriptive and quantitative studies by electron microscopy suggested a more advanced development of DMD myotubes as compared to normal ones. This conclusion was supported by the significantly larger capacitance of the DMD myotubes (408 +/- 45 pF) than of the normal myotubes (299 +/- 34 pF) of the same apparent size. 7. Taken together, these results show that differences in T- and L-type calcium currents between normal and DMD myotubes cannot simply explain all observed alterations in calcium homeostasis in DMD myotubes, thus suggesting that other transmembrane calcium transport mechanisms must also be altered in DMD myotubes compared with normal myotubes.

Cationic channels in normal and dystrophic human myotubes.

Human skeletal muscle cells obtained from normal and Duchenne muscular dystrophy patients were cocultured with explants of rat dorsal root ganglions. Single-channel recordings were performed with the cell-attached configuration of the patch-clamp technique and negative pressure was applied via the patch-pipette in order to mechanically stimulate the membrane patch. Inward elementary current activity was recorded under control or negative pressure conditions. Its occurrence and mean open probability were higher in Duchenne muscular dystrophy. Amplitude histograms reveal that these channels have a small unitary conductance of around 10 pS in 110 mM Ca2+ and could be inhibited in a dose-dependent manner by gadolinium. Results show that the membrane stress favoured calcium permeation through these channels. Taken together these data provide arguments for the involvement of such channels in calcium overload previously observed in cocultured dystrophic human (Duchenne muscular dystrophy) muscle cells.

Calcium homeostasis and cell death in Sol8 dystrophin-deficient cell line in culture.

Abnormalities of calcium homeostasis are involved in the process of cell injuries such as Duchenne muscular dystrophy characterized by the absence of the protein dystrophin. But how the absence of dystrophin leads to cytosolic calcium overload is as yet poorly understood. This question has been addressed with skeletal muscle cells from human DMD muscles or mdx mice. Although easier to obtain than human muscles, mdx muscle cells have provided controversial data concerning the resting intracellular calcium level ([Ca2+](i)). This work describes the culture of Sol8 cell line that expresses neither dystrophin nor adhalin, a dystrophin-associated protein. The [Ca2+](i)and intracellular calcium transients induced by different stimuli (acetylcholine, caffeine and high potassium) are normal during the first days of culture. At later stages, calcium homeostasis exhibits drastic alterations with a breaking down of the calcium responses and a large [Ca2+](i)elevation. Concomitantly, Sol8 cells exhibit morphological signs of cell death like cytoplasmic shrinkage and incorporation of propidium iodide. Cell death could be significantly reduced by blocking the activity of calpains, a type of calcium-regulated proteases. These results suggest that Sol8 cell line provides an alternative model of dystrophin-deficient skeletal muscle cells for which a clear disturbance of the calcium homeostasis is observed in culture in association with calpain-dependent cell death. It is shown that transfection with a plasmid cDNA permits the forced expression of dystrophin in Sol8 myotubes as well as a correct sorting of the protein. This approach could be used to explore possible interactions between dystrophin deficiency, calcium homeostasis alteration, and dystrophic cell death.

The effect of methylprednisolone on intracellular calcium of normal and dystrophic human skeletal muscle cells.

Clinical trials have shown that a glucocorticoid, the methyiprednisolone (PDN), has a beneficial effect on muscle strength and function in Duchenne muscular dystrophy (DMD) patients. The aim of this study was to test if the effect of PDN could be mediated via a possible action on intracellular calcium. The intracellular calcium activity, at rest and during calcium mobilizing drug superfusion protocols was recorded in normal and dystrophic human cocultured muscle cells. PDN (10 microM) pretreatment induced an elevation of the resting calcium concentration of 51, 34 and 38% in proliferating normal myoblasts, DMD myoblasts and DMD myotubes, respectively, while normal myotubes resting [Ca2+]i was not altered.

Hypoosmotic shocks induce elevation of resting calcium level in Duchenne muscular dystrophy myotubes contracting in vitro.

In Duchenne muscular dystrophy (DMD) muscle cells which lack dystrophin, contraction seems to be a dominant factor contributing to the abnormal elevated intracellular calcium level. Human normal and DMD contracting myotubes cocultured with nervous cells were exposed to a hypotonic medium to mimic contraction-induced mechanical stress on the membrane, and the cytoplasmic calcium activity was simultaneously monitored (Indo-1). Hypotonic shocks induced a reversible [Ca2+]i increase in 81% of the DMD cells vs. 54% of control. In addition, responses were qualitatively different: most of DMD myotubes displayed a fast increase of Ca2+ flowing from the edge of the myotube while the response in normal cells was slow and diffuse. The fact that these responses were not affected by ryanodine, was in favour of an external source of Ca2+ involved in the hypoosmotic shocks. The localized increase of Ca2+ in DMD myotubes, inhibited by Gd3+, could result from sites of high mechanosensitive channel activity or density which could constitute a pathway for Ca2+ entry provided these cells contract.