PubMed Health⌕ Search

Biomedical subjects

D Escande

Publications and source records attributed to D Escande.

At least 55 records · Page 3Linked to original sources
PubMed1997

KvLQT1 potassium channel but not IsK is the molecular target for trans-6-cyano-4-(N-ethylsulfonyl-N-methylamino)-3-hydroxy-2,2-dimethyl- chromane.

Mutations in the KvLQT1 gene are the cause for the long QT syndrome [Circulation 94:1996-2012 (1996)]. Coexpression of KvLQT1 in association with the channel regulator protein IsK produces a K+ current with characteristics reminiscent of the slow component of the delayed rectifier in cardiac myocytes. We explored the pharmacological properties of trans-6-cyano-4-(N-ethylsulfonyl-N-methylamino)-3-hydroxy-2,2-dime thyl- chromane (293B), a chromanol compound, on the K+ current produced by direct intranuclear injection of KvLQT1 and IsK cDNA plasmids in COS-7 cells. Injected cells were recorded by means of the whole-cell and cell-attached patch-clamp configurations under chloride-free conditions. Cells injected with KvLQT1 cDNA alone exhibited a fast-activating outward K+ current, whereas cells coinjected with KvLQT1 plus IsK cDNAs exhibited a time-dependent outward current with slower activation kinetics. The chromanol 293B blocked the K+ current related to KvLQT1 expression in both the absence or presence of IsK. The IC50 value for 293B to block KvLQT1-related current was not significantly modified by the presence of IsK (9.9 microM in the absence of IsK versus 9.8 microM in its presence). The block produced by 293B was strongly voltage-dependent inasmuch as it was close to 0 at -80 mV and occurred during a depolarizing voltage step. The time constants for the drug to block the current were in the same order of magnitude as activation kinetics of the current. Kinetics for drug unblock at the holding potential were much faster, in the order of a few tenths of a msec. KvLQT1 currents recorded in the cell-attached configuration were also blocked by externally applied 293B, suggesting that the compound penetrated the cell to block the channel. Cromakalim, another chromanol compound, also blocked KvLQT1 currents. Our results show that the chromanol compound 293B is targeted to KvLQT1 channels but not to the IsK regulator.

Animals↗
PubMed1996

Expression of CFTR controls cAMP-dependent activation of epithelial K+ currents.

The perforated-patch configuration of the patch-clamp technique was used to record whole cell currents from human epithelial CFPAC-1 cells defective for functional cystic fibrosis transmembrane conductance regulator (CFTR). In CFPAC-1 cells, adenosine 3',5'-cyclic monophosphate (cAMP) stimulation with forskolin (10 microM) plus 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (400 microM) activated neither Cl- nor K+ currents. In the same cells transfected with wild-type CFTR gene, cAMP stimulation produced activation of both Cl- and K+ currents. In Cl(-)-depleted medium (gluconate as a substitute), cAMP stimulation evoked a K+ current in CFTR-transfected but not in untransfected CFPAC-1 cells. This cAMP-evoked K+ current was the sum of two components: 1) a time-independent inwardly rectifying component, and 2) a slowly relaxing component activated at positive voltages. Increasing intracellular Ca2+ with ionomycin (1 microM) activated K+ currents in either transfected or untransfected cells. In transfected cells, blocking the CFTR conductance with high-concentration glibenclamide (100 microM) reduced the K+ current when activated by cAMP but not when activated by Ca2+. Pretreating CFTR-transfected cells for 48 h with interferon-gamma downregulated CFTR gene expression and reduced cAMP but not Ca2+ activation of the whole cell K+ current. From these results, we conclude that functional membrane CFTR protein influences activation by cAMP of epithelial K+ currents.

Calcium↗
PubMed1996

Adenosine A1 stimulation activates delta-protein kinase C in rat ventricular myocytes.

By making use of immunoblotting and immunocytochemical analysis, we explored whether stimulation of adenosine A1 receptors would promote the activation of delta-protein kinase C (delta-PKC) immunolabeled with a polyclonal antibody. Immunoblot analysis of Triton X-100-soluble cell membrane and cytosolic fractions revealed the presence of a specific 75-kD band reactive to the delta-PKC polyclonal antibody. In freshly isolated rat cardiac myocytes, 28% of the total immunoreactive delta-PKC was associated with the membrane fraction, whereas 72% was associated with the soluble fraction. Under stimulation with the tumor-promoting phorbol 12-myristate 13-acetate (PMA, 50 nmol/L) used as a positive control, delta-PKC translocated to the cell membrane, with the membrane fraction representing 88% and the cytosolic fraction representing 12% of the total immunoreactive delta-PKC. Transverse optical sections performed with confocal laser microscopy showed that immunostaining with anti-delta-PKC antibody was distributed in the cytosol membrane under PMA stimulation. In the membrane fraction of cells pretreated with adenosine (100 mumol/L) or with the adenosine A1 agonist (--)-N6-(2-phenylisopropyl)-adenosine (R-PIA, 1 mumol/L), the 75-kD band corresponding to delta-PKC increased by 57% and 66%, respectively, when compared with nonstimulated cells processed under the same experimental conditions. In cells exposed to either of the purine agonists, specific fluorescence staining decorated the cell membrane, a pattern that was not observed in control cells. Activation of membrane delta-PKC produced either by adenosine itself or by its analogue R-PIA was fully antagonized by the specific A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (1 mumol/L). From these data, we conclude that adenosine A1 stimulation activates delta-PKC in freshly isolated rat ventricular myocytes.

Adenosine↗
PubMed1996

A method for the rapid detection of recombinant CFTR during gene therapy in cystic fibrosis.

We developed an assay to detect wild-type CFTR in respiratory epithelial cells with the objective to evaluate the efficacy of DNA delivery during in vivo gene transfer. The method is based on the previous observation that the common delta F508-CFTR mutant does not reach the apical membrane as does the transgene product. We thus used a monoclonal antibody, MATG 1031, raised against the first extracellular loop sequence of the CFTR protein and an immunodetection protocol lacking premature fixation or permeabilization. Specificity of MATG 1031 for its epitope was controlled by immunoblotting. In HT29-19A, 184, CAPAN-1 human cell lines, and in respiratory primary cultures, staining with MATG 1031, examined by confocal scanning laser microscopy, appeared as small dots restricted to the apical surface. No such staining was observed in NIH-3T3 fibroblasts, in the cystic fibrosis cell line CFPAC-1 or in primary cultures from cystic fibrosis patients. Apical immunostaining with MATG 1031 was restored in CFPAC-1 cells cultured at a low temperature (30 degrees C) and in CFPAC-1 cells transfected with wild-type CFTR Recombinant CFTR was also recognized in CF respiratory cells lipotransfected with wild-type CFTR plasmid DNA MATG 1031 immunostaining was further investigated under blinded conditions in primary cultures derived from nasal curettage. In all the cell cultures examined, our protocol allowed discrimination between non-CF and CF cells. We propose that this method is convenient to detect apical CFTR and may be used to monitor in vivo gene transfer.

3T3 Cells↗
PubMed1996

Acute simulated ischaemia produces both inhibition and activation of K+ currents in isolated ventricular myocytes.

OBJECTIVE: The aim was to investigate the effects of acute ischaemia on cardiac repolarizing K+ currents. METHODS: We developed a model of acute ischaemia in isolated rat ventricular myocytes transiently surrounded with a mineral oil droplet. During ischaemic challenges, we recorded intracellular pH using the fluorescent probe seminaphthorhodafluor-1 (SNARF-1) and whole-cell K+ currents using the patch-clamp technique. RESULTS: Decrease in intracellular pH (pH1) during simulated ischaemia was dependent upon the extracellular proton buffer used (pH1 decreased from 7.44 +/- 0.02 to 7.16 +/- 0.04 in a Hepes-buffered medium and from 7.08 +/- 0.04 to 6.56 +/- 0.07 with bicarbonate buffer). In Hepes, action potential duration initially lengthened and then shortened under the effects of ischaemia. Initial action potential duration lengthening was concomitant with a block of the inward rectifier K+ current, whereas late shortening corresponded with the activation of the ATP-sensitive K+ current. Similar changes occurred in bicarbonate buffer although with different amplitudes and kinetics. Patch-clamp experiments also showed inhibition of the transient outward K+ current. Brief transient episodes of ischaemia activated ATP-sensitive K+ current in only 20% of control cells (n = 21) but in 100% of cells treated with 15 microM cromakalim (n = 9). CONCLUSIONS: (i) Simulated ischaemia produces complex effects on repolarizing K+ currents including both inhibition and activation; (ii) cromakalim accelerates activation of ATP-sensitive K+ current during simulated ischaemia.

Action Potentials↗
PubMed1995

ATP-sensitive K+ channels regulated by intracellular Ca2+ and phosphorylation in normal (T84) and cystic fibrosis (CFPAC-1) epithelial cells.

The elementary K+ conductance activated by the cAMP or the Ca2+ second messenger pathways was investigated in the model salt-secreting epithelium, the human T84 cell line. Under Cl(-)-free conditions, an inwardly rectifying whole-cell K+ current was evoked by either forskolin 10 (mumol/l) or acetylcholine 1 (mumol/l) and blocked by extracellular charybdotoxin 10 (nmol/l). In the cell-attached mode, both secretory agonists induced the opening of a channel showing inward rectification with a unitary chord conductance of 36.8 +/- 2.5 pS (n = 26) for inward currents. In inside-out patches, a 35-pS inwardly rectifying K+ channel that corresponded to the channel recorded in the cell-attached configuration was recorded in the presence of 0.3 mumol/l free Ca2+ at the inner side of the membrane. This channel was blocked by Ba2+ (5 mumol/l) and by charybdotoxin (50 nmol/l). Its open probability was enhanced by intracellular Ca2+ with and EC50 of 0.25 mumol/l and strongly reduced by intracellular MgATP with an IC50 of 600 mumol/l. In the continuous presence of ATP, the channel activity was consistently increased by 125 kU/l catalytic subunit of cAMP-dependent protein kinase. In the cystic fibrosis pancreatic duct cell line CFPAC-1, a K+ channel was also recorded, with similar characteristics and regulation as the 35-pS channel in T84 cells. We conclude that an ATP-sensitive K+ channel regulated by intracellular Ca2+ and phosphorylation supports the main K+ current activated by secretory agonists in normal cystic fibrosis cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1995

[Pharmacology of ion channels in mucoviscidosis. Physiological bases and therapeutic applications].

Phenotypical expression of cystic fibrosis (CF) includes decreased epithelial chloride secretion and increased sodium absorption. These anomalies produce increased water absorption and a dehydrated mucus responsible for decreased mucociliary clearance. Identification of the gene responsible for the genetic disease (CFTR for cystic fibrosis transmembrane conductance regulator) together with a more accurate comprehension of complexes interactions that exist between the CFTR gene product and other epithelial ionic channels has created novel opportunities for discovering specific pharmacological drugs to treat the disease. Amiloride, which limits sodium hyperabsorption, has demonstrated both efficacy and safety in vivo in a restricted number of adult patients. Nucleotides such as ATP or UTP, prescribed in association with amiloride, increase chloride secretion. Potassium channel openers, by stimulating transepithelial chloride transport, may represent an additional innovative approach. Specific pharmacology to CF is not competitive but rather complementary to gene therapy.

Adenosine Triphosphate↗
PubMed1995

[Ischemic preconditioning: concept of endogenous myocardial protection].

Preconditioning is a temporary tolerance to ischaemia acquired by the myocardium after a short period of ischaemia. It results in the limitation of the infarct size induced by prolonged coronary occlusion. The mechanism of this cytoprotection remains poorly understood. The A1 adenosine receptors, the ATP-sensitive potassium channels and protein-kinase C seem to play prominent roles. The effects of preconditioning on the complications of ischaemia/reperfusion such as myocardial stunning, ventricular arrhythmias or decreased coronary reserve are not well known. Several studies suggest that the cytoprotection resulting from preconditioning could be applied to human myocardium and constitute a preventive anti-ischaemic therapy during coronary angioplasty, cardiac surgery or the conservation of transplant grafts.

Adaptation, Physiological↗
PubMed1994

Concomitant activation of Cl- and K+ currents by secretory stimulation in human epithelial cells.

1. Whole-cell currents were investigated in the model salt-secreting epithelium, human T84 cell line, by means of the perforated patch-clamp technique. In the control extracellular medium containing Cl-, depolarizing voltage ramps evoked current responses which peaked at 5.43 +/- 0.81 pA pF-1 at +60 mV and had a reversal potential (Erev) of -38.4 +/- 2.5 mV (n = 23). 2. Activation of the cAMP pathway with forskolin increased the current at +60 mV from 3.81 +/- 0.61 to 20.79 +/- 5.08 pA pF-1 (n = 18). In thirteen cells, Erev was initially shifted towards positive potentials (Erev of the cAMP-activated initial current was -18.2 +/- 1.2 mV) and subsequently shifted towards more negative potentials, consistent with the activation of both Cl- and K+ currents during cAMP stimulation. 3. Increasing the intracellular Ca2+ concentration, [Ca2+]i, with ionomycin (1 microM) or with acetylcholine (1 microM), increased the current at +60 mV from 7.79 +/- 1.57 to 57.50 +/- 12.10 pA pF-1 (n = 6) and from 6.36 to 34.13 pA pF-1 (n = 4), respectively. With both agonists, Erev was shifted either towards the reversal potential for potassium, EK, or towards the reversal potential for chloride, ECl, depending on the cell. 4. In the absence of chloride ions (gluconate substituted), stimulation of the Ca2+ pathway activated a time-independent outward current of large amplitude. This current exhibited inward rectification at positive voltages, reverted at -89.5 +/- 0.2 mV and was markedly reduced by charybdotoxin (10 nM), a specific blocker of Ca(2+)-activated K+ channels. When a voltage step protocol was used, increased [Ca2+]i also activated an outward current at potentials more positive than -40 mV which slowly relaxed during depolarizing steps. 5. The activation of both (i) a time-dependent inwardly rectifying charybdotoxin-sensitive K+ current, and (ii) a time-dependent slowly inactivating current was also produced by cAMP stimulation. 6. We concluded that (i) in the T84 epithelial cells, both Cl- and K+ currents are concomitantly increased by secretagogue stimulation, and (ii) two different types of K+ conductances are activated by either the cAMP or the intracellular Ca2+ secreting pathways.

Acetylcholine↗
PubMed1994

Effects of sulphonylureas on cAMP-stimulated Cl- transport via the cystic fibrosis gene product in human epithelial cells.

The cystic fibrosis gene product (CFTR) is a Cl- channel that possesses specific binding sites for cytosolic adenosine triphosphate (ATP) and is activated by cyclic adenosine monophosphate (cAMP)-dependent protein kinases. We explored the possibility that CFTR shares a common pharmacology with another ATP-regulated channel protein, the ATP-sensitive K+ channel that is blocked by sulphonylureas and activated by diazoxide. cAMP-stimulated Cl- effluxes were measured with 36Cl- in the epithelial cell line T84 which stably expresses CFTR. Neither glibenclamide (30 microM), tolbutamide (1 mM) nor diazoxide (100 microM) significantly affected forskolin-activated 36Cl- effluxes in T84 cells. In patch-clamp experiments, glibenclamide exerted only weak inhibitory effects on the whole-cell currents through CFTR with an IC50 of around 0.1 mM. Tolbutamide at 1 mM, but not at 0.1 mM, blocked a current of small amplitude which reversed near the equilibrium potential for K+ ions. We conclude that sulphonylureas and diazoxide are not effective antagonists of endogenous CFTR Cl- channels.

Adenosine Triphosphate↗
PubMed1994

Do potassium channel openers compete with ATP to activate ATP sensitive potassium channels?

Potassium channel openers are a class of chemically heterogeneous compounds that produce vasodilatation by opening vascular smooth muscle potassium channels. Using various configurations of the powerful patch clamp technique, the molecular pharmacology of potassium channel openers has been largely explored in different cellular systems. These studies have revealed that the primary molecular target for potassium channel openers is this class of potassium channel proteins that close under the influence of intracellular ATP and have thus been named ATP sensitive potassium channels. Direct recordings of unitary ATP sensitive potassium channels in cell detached membrane patches support the view that potassium channel openers compete with intracellular ATP to prevent channel inhibition. However, there also exists experimental evidence that contradicts the competition hypothesis and suggests the involvement of phosphorylation-dephosphorylation mechanisms. The aim of the present article is to review experimental data that support, or inversely, contradict the potassium channel opener-ATP competition theory.

Adenosine Triphosphate↗
PubMed1994

Interferon-gamma downregulates CFTR gene expression in epithelial cells.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in defective transepithelial Cl- transport. The regulation of CF gene expression is not fully understood. We report that interferon-gamma (IFN-gamma), but not IFN-alpha or -beta, downregulates CFTR mRNA levels in two colon-derived epithelial cell lines, HT-29 and T84, in a time- and concentration (from 0.1 IU/ml)-dependent manner. IFN-gamma has no effect on the transcription rate of the CFTR gene but reduces CFTR mRNA half-life, indicating that it exerts a posttranscriptional regulation of CFTR expression, at least partly, through destabilization of the transcripts. Cells treated with IFN-gamma contain subnormal amounts of 165-kDa CFTR protein. Assays of adenosine 3',5'-cyclic monophosphate-stimulated 36Cl- efflux and whole cell currents show that CFTR function is diminished in IFN-gamma-treated cells. IFN-gamma and tumor necrosis factor-alpha synergistically reduce CFTR gene expression. Our results suggest that production of these cytokines in response to bacterial infections and inflammatory disorders may alter transmembrane Cl- transport.

Cell Membrane Permeability↗
PubMed1994

Abnormal subcellular localization of mutated CFTR protein in a cystic fibrosis epithelial cell line.

The cystic fibrosis gene product, CFTR, is a Cl- channel that possesses specific binding sites for cytosolic ATP and is activated by cAMP-dependent protein kinase. Most recently, it was reported that CFTR localizes at the surface apical compartment of normal airway epithelial cells, but accumulates in the cytosol of airway cells from CF patients with the delta F508 mutation. In order to explore whether the same difference exists in normal and CF established cell lines that are commonly used in physiological and pharmacological investigations of the CF defect, we employed monoclonal antibodies raised against synthetic peptides corresponding to two different regions of the CFTR protein. One antibody (MATG 1061) was generated against amino acids 503-515 delta 508 in the nucleotide binding domain 1, whereas the other (MATG 1031) was generated against amino acids 107-117 situated in a putative external loop. We used confocal laser scanning microscopy to localize the CFTR protein in T84 (a colonic derived carcinoma), CAPAN-1 (a pancreatic carcinoma), and in CFPAC-1 (a pancreatic carcinoma homozygous for the delta F508 deletion) cell lines. In permeabilized T84 and CAPAN-1 cells, immunolabeling with MATG 1061 predominated at the apical domain. By contrast, CFTR staining with MATG 1061 was homogeneously distributed in the cytoplasm of CFPAC-1 cells. In non-permeabilized non-CF cell lines, MATG 1031 specifically labeled an apical membrane surface epitope. No such labeling was present in CFPAC-1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Line↗
PubMed1994

[Positive inotropic and lusitropic effect of RP 62719, a new class III antiarrhythmia agent].

Antiarrhythmic drugs, especially the Class I family, exert a negative inotropic effect on the myocardium which is particularly undesirable in patients with depressed left ventricular function. Therefore, research has been directed to the development of new, more specific molecules of the Class III family. The authors studies the mechanical effects of RP 62719 on guinea pig left ventricular papillary muscle. This new molecule is a pure Class III antiarrhythmic, known to lengthen the duration of the cardiac action potential by selectively blocking the potassium current iK1 (inward rectifier K+ current). The mechanical parameters were determined during the phases of contraction and relaxation under isotonic and isometric conditions. At 0.2 and 2 microM concentrations, RP 62719 improved cardiac contraction under both isotonic and isometric conditions with an increase of about 30% of Vmax (p < 0.001), the maximum unloaded shortening velocity delta 1 (p < 0.001), the peak isometric active force normalized per cross-sectional area [AF/S (p < 0.001)]. At these two concentrations, a positive lusitropic effect (improved relaxation) was demonstrated by an increase in negative peak of derivative per mm2-dF/s and maximum lengthening velocity VR max (p < 0.01). At higher concentrations (20 microM), the inotropic and lusitropic effects were less marked with a bell-shaped form of the dose-effect curve. This study indicates that RP 62719 has moderate but significant positive inotropic and lusitropic effects. These actions could provide significant therapeutic advantages especially in patients cardiac failure.

Animals↗
PubMed1993

Fast K channels are more sensitive to riluzole than slow K channels in myelinated nerve fibre.

The effects of 1-500 microM riluzole, a novel psychotropic agent, were studied on the nodal K current of isolated nerve fibres of the frog. When added to the external solution, the substance rapidly and reversibly inhibited slow, fast 1 and fast 2 K components of the tail K current. The concentrations of riluzole inducing half maximum reduction of slow, fast 1 and fast 2 K conductances were 413 microM, 24 microM and 21 microM respectively. It is concluded that the substance is about 20 times more effective in blocking fast than slow K channels.

Anesthetics↗