Biomedical subjects

Alicia E Damiano

Publications and source records attributed to Alicia E Damiano.

3 recordsLinked to original sources

PubMed2005

Ionic currents in multidrug resistant K562 human leukemic cells.

In this study, the expression and functional characterization of currents through the CFTR (cystic fibrosis transmembrane regulator) and ORCC (outwardly rectifying chloride channels) were determined in wild-type K562 chronic human leukemia cells (K562-WT) and in its resistant counterpart, the vincristine resistant cell line (K562-Vinc). Expression of the CFTR and MDR1 (multidrug resistant) gene products was determined by a semi-quantitative RT-PCR protocol. The amplified products in K562-WT and K562-Vinc showed two bands corresponding to CFTR and MDR1. MDR1 mRNA increased by 20-fold in K562-Vinc whereas no change in CFTR mRNA levels was observed. CFTR and ORCC channel activity were measured with a whole cell configuration of the patch clamp technique. Forskolin (40 microM n activator of adenylate cyclase, added to the extracellular side increased the current in both cell lines. A fraction of the activated whole cell currents was inhibited by 500 microM 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) and subsequent addition of 500 microM diphenylamine-2-carboxylate (DPC plus DIDS) further inhibited the remaining currents. The levels of forskolin-activated currents and subsequent blockade were similar in both cell lines. The effect of forskolin was prevented in cells previously exposed to 500 microM DPC. The effects of DIDS and DPC on the forskolin-activated whole cell currents support the idea that both CFTR and ORCC are generating a significant fraction of these currents with DIDS inhibiting ORCC currents and DPC inhibiting CFTR currents when the blockers are added one after another to the extracellular side. Finally, we show that exposure of K562 cells to vincristine which results in the over expression of MDR1 is not accompanied by a significant down regulation of CFTR as in other cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2003

CFTR in K562 human leukemic cells.

In this study, the expression and functional characterization of CFTR (cystic fibrosis transmembrane regulator) was determined in K562 chronic human leukemia cells. Expression of the CFTR gene product was determined by RT-PCR and confirmed by immunohistochemistry and Western blot analysis. Functional characterization of CFTR Cl- channel activity was conducted with patch-clamp techniques. Forskolin, an adenylyl cyclase activator, induced an anion-selective channel with a linear current-voltage relationship and a single-channel conductance of 11 pS. This cAMP-activated channel had a Pgluconate/PCl or PF/PCl perm-selectivity ratio of 0.35 and 0.30, respectively, and was inhibited by the CFTR blocker glibenclamide and the anti-CFTR antibody MAb 13-1, when added to the cytoplasmatic side of the patch. Glibenclamide decreased the open probability increasing the frequency of open-to-closed transitions. Addition of 200 microM DIDS caused an irreversible block of the channels when added to the cytosolic side of inside-out patches. These and other observations indicate a widespread distribution of CFTR gene expression and suggest that this channel protein may function in most human cells to help maintain cellular homeostasis.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2002

Membrane currents in the oocyte of the toad Bufo arenarum.

The amphibian oocyte cell model is widely used for heterologous expression of ionic channels and receptors. Little is known, however, about the physiology of oocyte cell models other than Xenopus laevis. In this study, the two-electrode voltage clamp technique was used to assess the most common electrical patterns of oocytes of the South American toad Bufo arenarum. Basal membrane resistance, resting potential, and ionic currents were determined in this cell model. The oocyte transmembrane resistance was 0.35 M(Omega), and the resting potential in normal saline was about -33 mV with a range between -20 mV and -50 mV. This is, to our knowledge, the first attempt to begin an understanding of the ion transport mechanisms of Bufo arenarum oocytes. This cell model may provide a viable alternative to the expression of ion channels, in particular those endogenously observed in Xenopus laevis oocytes.

Action Potentials↗