S-CMC-Lys protective effects on human respiratory cells during oxidative stress.

The mucoactive drug S-carbocysteine lysine salt monohydrate (S-CMC-Lys) stimulates glutathione (GSH) efflux from respiratory cells. Since GSH is one of the most important redox regulatory mechanisms, the aim of this study was to evaluate the S-CMC-Lys effects on GSH efflux and intracellular concentration during an oxidative stress induced by the hydroxyl radical (xOH). Experiments were performed on cultured human respiratory WI-26VA4 cells by means of patch-clamp experiments in whole-cell configuration and of fluorimetric analyses at confocal microscope. xOH exposure induced an irreversible inhibition of the GSH and chloride currents that was prevented if the cells were incubated with S-CMC-Lys. In this instance, the currents were inhibited by the specific blocker CFTR(inh)-172. CFT1-C2 cells, which lack a functional CFTR channel, were not responsive to S-CMC-Lys, but the stimulatory effect of the drug was restored in LCFSN-infected CFT1 cells, functionally corrected to express CFTR. Fluorimetric measurements performed on the S-CMC-Lys-incubated cells revealed a significant increase of the GSH concentration that was completely hindered after oxidative stress and abolished by CFTR(inh)-172. The cellular content of reactive oxygen species was significantly lower in the S-CMC-Lys-treated cells either before or after xOH exposure. As a conclusion, S-CMC-Lys could exert a protective function during oxidative stress, therefore preventing or reducing the ROS-mediated inflammatory response.

Volume-regulated Cl- channels in human pleural mesothelioma cells.

Anion channels in human mesothelial and mesothelioma cell lines were characterized by patch-clamp and biomolecular approaches. We found an outwardly rectifying anionic current which was inactivated at positive voltages and inhibited by extracellular adenosine 5'-triphosphate (ATP). Mesothelial and mesothelioma cells behaved differently concerning current inactivation properties. Inactivation is more pronounced and has a steeper onset in mesothelial cells. Different reversal potentials, in asymmetrical Cl(-) solutions, that could be attributed to a different selectivity of the channel, have been observed in the two cell lines. Mesothelioma cell single-channel analysis indicates that the number of the same active anion channel (3-4 pS) increased under hypoosmotic conditions. Immunocytochemistry experiments showed the presence of ICln protein in the cytosol and in the plasma membrane. Western blot analysis revealed an increase of ICln in the membrane under hypotonic conditions, an event possibly related to the activation of Cl(-) channels.

A new gene-finding tool: using the Caenorhabditis elegans operons for identifying functional partner proteins in human cells.

How can a large number of different phenotypes be generated by a limited number of genotypes? Promiscuity between different, structurally related and/or unrelated proteins seems to provide a plausible explanation to this pertinent question. Strategies able to predict such functional interrelations between different proteins are important to restrict the number of putative candidate proteins, which can then be subjected to time-consuming functional tests. Here we describe the use of the operon structure of the nematode genome to identify partner proteins in human cells. In this work we focus on ion channels proteins, which build an interface between the cell and the outside world and are responsible for a growing number of diseases in humans. However, the proposed strategy for the partner protein quest is not restricted to this scientific area but can be adopted in virtually every field of human biology where protein-protein interactions are assumed.

K+ channel cAMP activated in guinea pig gallbladder epithelial cells.

In guinea pig gallbladder epithelial cells, an increase in intracellular cAMP levels elicits the rise of anion channel activity. We investigated by patch-clamp techniques whether K(+) channels were also activated. In a cell-attached configuration and in the presence of theophylline and forskolin or 8-Br-cAMP in the cellular incubation bath, an increase of the open probability (P(o)) values for Ca(2+)-activated K(+) channels with a single-channel conductance of about 160 pS, for inward current, was observed. The increase in P(o) of these channels was also seen in an inside-out configuration and in the presence of PKA, ATP, and cAMP, but not with cAMP alone; phosphorylation did not influence single-channel conductance. In the inside-out configuration, the opioid loperamide (10(-5) M) was able to reduce P(o) when it was present either in the microelectrode filling solution or on the cytoplasmic side. Detection in the epithelial cells by RT-PCR of the mRNA corresponding to the alpha subunit of large-conductance Ca(2+)-activated K(+) channels (BK(Ca)) indicates that this gallbladder channel could belong to the BK family. Immunohistochemistry experiments confirm that these cells express the BK alpha subunit, which is located on the apical membrane. Other K(+) channels with lower conductance (40 pS) were not activated either by 8-Br-cAMP (cell-attached) or by PKA + ATP + cAMP (inside-out). These channels were insensitive to TEA(+) and loperamide. The data demonstrate that under conditions that induce secretion, phosphorylation activates anion channels as well as Ca(2+)-dependent, loperamide-sensitive K(+) channels present on the apical membrane.