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Biomedical subjects

Aleksander Edelman

Publications and source records attributed to Aleksander Edelman.

27 records · Page 2Linked to original sources
PubMed2004

Microelectrodes and their use to assess ion channel function.

Impalement of living cells with microelectrodes (MEs) is a useful approach to measure a variety of biological parameters such as membrane potential (Vm), intracellular free ion concentrations and cell-to-cell communication. Despite such widespread applications, the number of trained researchers capable of using MEs is diminishing. In this article, we describe the fundamentals of the preparation and the proper use of MEs to study the physiology of transepithelial ion transport.

Animals↗
PubMed2004

Basic protocol for transepithelial nasal potential difference measurements.

Transepithelial nasal potential difference (NPD) measurements assess ion conductance in the upper respiratory epithelium. NPD is useful in assisting in the diagnosis of classical and atypical cystic fibrosis (CF) and of cystic fibrosis transmembrane regulator (CFTR)-related disorders, as well as for monitoring the effect of pharmacological agents and gene transfer approaches to correct the abnormalities of ion transport in CF. The article summarizes the objectives and the principle of NPD measurements, describes a hands-on protocol of the procedure and provides quality control measures, practical hints and troubleshooting.

Clinical Protocols↗
PubMed2004

Measurement of halide efflux from cultured and primary airway epithelial cells using fluorescence indicators.

The use of the halide-sensitive fluorescent probes (6-methoxy-N-(-sulphopropyl)quinolinium (SPQ) and N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE)) to measure chloride transport in cells has now been established as an alternative to the halide-selective electrode technique, radioisotope efflux assays and patch-clamp electrophysiology. We report here procedures for the assessment of halide efflux, using SPQ/MQAE halide-sensitive fluorescent indicators, from both adherent cultured epithelial cells and freshly obtained primary human airway epithelial cells. The procedure describes the calculation of efflux rate constants using experimentally derived SPQ/MQAE fluorescence intensities and empirically derived Stern-Volmer calibration constants. These fluorescence methods permit the quantitative analysis of CFTR function.

Cells, Cultured↗
PubMed2003

Evaluation of MRP1-5 gene expression in cystic fibrosis patients homozygous for the delta F508 mutation.

Cystic fibrosis (CF), due to mutations of the cystic fibrosis transmembrane conductance regulator (CFTR), exhibits a wide range of disease severity, even among deltaF508 homozygous patients, and the mechanisms of this variability have yet to be elucidated. In view of the close structural homology and possible functional overlap between CFTR and Multidrug Resistance-associated Proteins (MRPs), MRPs were investigated as potentially relevant factors in CF pathophysiology. MRP1-5 gene expression was analyzed in nasal respiratory epithelial cells from deltaF508 homozygous patients (n = 19) and control subjects (n = 20) using semiquantitative RT-PCR. Significantly lower MRP1 and MRP5 transcript levels were found in CF patients than in control subjects. MRP1 and MRP5 transcript levels were strongly correlated (r = 0.71). In CF patients, low MRP1 transcript levels were associated with more severe disease as assessed by the Shwachman score. A relation was also observed between MRP1 levels and presence of a cAMP-independent chloride conductive pathway, as determined by a halide-sensitive fluorescent assay. These results suggest that MRPs, especially MRP1, might play a role in CF phenotype and might therefore constitute a target for a novel pharmacotherapy of CF.

Adolescent↗
PubMed2003

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. By altering or reproducing the ouabain-induced changes in intracellular ionic activities (decreasing in external Na(+) or K(+) or using Na(+) ionophore), we show that the ouabain-induced regulations of both CFTR and MDR-1 transcripts depend on the Na(+)/K(+) pump inhibition but that the decrease in CFTR mRNAs also proceeds from cytoplasm reactions simultaneously activated by ouabain. These data, which emphasize the complex mechanism of action of ouabain, suggest that changes in intracellular ionic activities modulate CFTR/MDR-1 gene expressions.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

The Gef1 protein of Saccharomyces cerevisiae is associated with chloride channel activity.

The Gef1 protein of the yeast Saccharomyces cerevisiae (Gef1p) has amino acid homology to the voltage-gated CLC chloride channel family. It has been postulated that it provides the compensatory transport of Cl- anions to the lumen of the Golgi thereby regulating the pH of this compartment. Using GEF1 fusion with heterologous promoter we obtained a yeast strain highly overproducing Gef1p. The electrophysiological properties of the microsomal fraction obtained from this strain were measured using lipid bilayer system. Our data indicate that Gef1p is associated with the chloride channel activity. This anion-selective channel has a unitary conductance of 42 pS when measured in symmetrical 600/600 mM TEA-Cl solutions, is voltage-dependent, and closes at high negative voltages.

Chloride Channels↗
PubMed2002

Distribution of ClC-2 chloride channel in rat and human epithelial tissues.

The ubiquitous ClC-2 Cl(-) channel is thought to contribute to epithelial Cl(-) secretion, but the distribution of the ClC-2 protein in human epithelia has not been investigated. We have studied the distribution of ClC-2 in adult human and rat intestine and airways by immunoblotting and confocal microscopy. In the rat, ClC-2 was present in the lateral membranes of villus enterocytes and was predominant at the basolateral membranes of luminal colon enterocytes. The expression pattern of ClC-2 in the human intestine differed significantly, because ClC-2 was mainly detected in a supranuclear compartment of colon cells. We found significant expression of ClC-2 at the apex of ciliated cells in both rat and human airways. These results show that the distribution of ClC-2 in airways is consistent with participation of ClC-2 channels in Cl(-) secretion and indicate that extrapolation of results from studies of ClC-2 function in rat intestine to human intestine is not straightforward.

Amino Acid Sequence↗
PubMed2002

Normal function of the cystic fibrosis conductance regulator protein can be associated with homozygous (Delta)F508 mutation.

Cystic fibrosis (CF) is caused by mutations of the gene encoding for the CFTR (CF transmembrane conductance regulator) protein. The most frequent mutation, the (Delta)F508 mutation, results in a defective cAMP-regulated chloride transport in the epithelial cells. The spectrum of clinical manifestations in patients bearing homozygous (Delta)F508 mutations can vary considerably, suggesting that, in the patients with a mild disease, CFTR could be partly functional. To test this hypothesis, we explored in nasal ciliated epithelial cells (NCC) of 9 control subjects and 23 (Delta)F508 homozygous patients the anion conductive pathway by a halide sensitive fluorescent dye assay SPQ (6-methoxy-N-3'-sulfopropylquinolinium) and the CFTR transcript levels by RT-PCR. As 50% represented the lowest fraction of the control subjects NCC demonstrating a cAMP-dependent conductance, a CF patient was considered as "cAMP responder" if at least 50% of the NCC tested displayed a cAMP-dependent conductive pathway. According to these criteria, 8 of the 23 patients were considered as cAMP responders. They had a significantly less severe disease considering the respiratory function and infectious status. The amount of CFTR mRNA did not differ between the control subjects and the patients. No statistical correlation could be found between the transcript level and the expression of a cAMP conductive pathway. This cAMP-dependent Cl(-) conductance detected in homozygous NCC could be due to a residual CFTR activity and may explain the mild phenotypes observed in some (Delta)F508 homozygous patients.

Chlorides↗