Long-chain acylcarnitines regulate the hERG channel.

BACKGROUND AND PURPOSE: In some pathological conditions carnitine concentration is high while in others it is low. In both cases,cardiac arrhythmias can occur and lead to sudden cardiac death. It has been proposed that in ischaemia, acylcarnitine (acyl-CAR), but not carnitine, is involved in arrhythmias through modulation of ionic currents. We studied the effects of acyl-CARs on hERG, K(IR)2.1 and K(v)7.1/minK channels (channels responsible for I(KR), I(K1) and I(KS) respectively). EXPERIMENTAL APPROACH: HEK293 cells stably expressing hERG, K(IR)2.1 or Kv7.1/minK were studied using the patch clamp technique. Free carnitine (CAR) and acyl-CAR derivatives from medium- (C8 and C10) and long-chain (C16 and C18:1) fatty acids were applied intra- and extracellularly at different concentrations. For studies on hERG, C16 and C18:1 free fatty acid were also used. KEY RESULTS: Extracellular long-chain (LCAC), but not medium-chain, acyl-CAR,induced an increase of I(hERG) amplitude associated with a dose-dependent speeding of deactivation kinetics. They had no effect on K(IR)2.1 or Kv7.1/minK currents.Computer simulations of these effects were consistent with changes in action potential profile. CONCLUSIONS AND APPLICATIONS: Extracellular LCAC tonically regulates I(hERG) amplitude and kinetics under physiological conditions. This modulation may contribute to the changes in action potential duration that precede cardiac arrhythmias in ischaemia, diabetes and primary systemic carnitine deficiency.

Altered SK3/KCa2.3-mediated migration in adenomatous polyposis coli (Apc) mutated mouse colon epithelial cells.

Lost of adenomatous polyposis coli gene (Apc) disturbs the migration of intestinal epithelial cells but the mechanisms have not been fully characterized. Since we have demonstrated that SK3/KCa2.3 channel promotes cancer cell migration, we hypothesized that Apc mutation may affect SK3/KCa2.3 channel-mediated colon epithelial cell motility. We report evidence that SK3/KCa2.3 channel promotes colon epithelial cells motility. Following Apc mutation SK3/KCa2.3 expression is largely reduced leading to a suppression of the SK3/KCa2.3 channel mediated-cell migration. Our findings reveal a previously unknown function of the SK3/KCa2.3 channel in epithelial colonic cells, and suggest that Apc is a powerful regulator SK3/KCa2.3 channel.

On the mechanisms of ultrasound contrast agents-induced arrhythmias.

Recent reports have shown that imaging hard-shelled ultrasound (US) contrast agents at high mechanical indices engenders premature ventricular contractions (PVCs). We have shown that the oscillations of microbubbles next to a cell induce a mechanical pressure on its membrane resulting in the activation of stretch activated channels (SAC). The aim of this study is to demonstrate, in vivo and in vitro, the relationship between PVCs and SAC opening. Five anesthetized rats were used. PVCs were created in vivo with (1) US and a diluted solution of contrast microbubbles injected intravenously through the tail vein at a rate of 0.5 mL per min and (2) a manually induced mechanical stimulus, which consisted of stimulations by a flexible catheter introduced into the rat aorta and pushed until the left ventricle. PVCs were quantified through ECG measurements. In vitro experiments consisted of patch Clamp measurements on HL-1 heart cell line. The stimulation was carried out either manually with a glass rod or with US and microbubbles. For both in vivo and in vitro experiments, US consisted of 40-cycle waveforms at 1 MHz and peak negative pressures up to 300 kPa and exposure time varied from 1 to 2 min. We should emphasize that these parameters are different from those used in diagnostic conditions. In vivo, microbubbles and US at 300 kPa induced modification of rat's ECG while pressures below 300 kPa did not induce any PVC. US alone did not modify the rat's ECG. Similar PVCs were also created when stimulation with a catheter was applied. Regular heart beat rate was recovered immediately after the stimulation was stopped. In vitro, the mechanical stretch induced a cell membrane depolarization due to SAC opening. Similar effect was observed with US and microbubbles. The cell potential returned to its initial value when the stimulation was released. In conclusion, we presume that PVCs are generated through a cascade of events characterized by a mechanical action of oscillating microbubbles, opening of stretch activated ion channels, membrane depolarization and triggering of action potentials.

Non-anti-mitotic concentrations of taxol reduce breast cancer cell invasiveness.

Taxol is widely used in breast cancer chemotherapy. Its effects are primarily attributed to its anti-mitotic activity. Microtubule perturbators also exert antimetastatic activities which cannot be explained solely by the inhibition of proliferation. Voltage-dependent sodium channels (Na(V)) are abnormally expressed in the highly metastatic breast cancer cell line MDA-MB-231 and not in MDA-MB-468 cell line. Inhibiting Na(V) activity with tetrodotoxin is responsible for an approximately 0.4-fold reduction of MDA-MB-231 cell invasiveness. In this study, we focused on the effect of a single, 2-h application of 10 nM taxol on the two cell lines MDA-MB-231 and MDA-MB-468. At this concentration, taxol had no effect on proliferation after 7 days and on migration in any cell line. However it led to a 40% reduction of transwell invasion of MDA-MB-231 cells. There was no additive effect when taxol and tetrodotoxin were simultaneously applied. Na(V) activity, as assessed by patch-clamp, indicates that it was changed by taxol pre-treatment. We conclude that taxol can exert anti-tumoral activities, in cells expressing Na(V), at low doses that have no effect on cell proliferation. This effect might be due to a modulation of signalling pathways involving sodium channels.

Characterization of cell membrane response to ultrasound activated microbubbles.

Contrast agents for ultrasound imaging, composed of tiny gas microbubbles, have become a reality in clinical routine. They are extensively used in radiology for detection and characterization of various tumors and in cardiology for left ventricular opacification. Recent experimental studies showed that ultrasound waves in combination with contrast agent microbubbles increase transiently cell membrane permeability in a process known as sonoporation. This effect is thought to allow foreign molecules to enter the cell. In that context, we explored the cell membrane's responses to microbubbles' oscillations as the mechanism is not completely understood. Breast cancer cell line in combination with contrast microbubbles were used. Ultrasound was applied using a transducer of 1 MHz center frequency transmitting a 10-cycle burst of different acoustic pressures repeated every 100 mus. Patch-clamp technique in whole cell configuration was used to explore transmembrane ion exchange through the variations in membrane potential. To characterize the activated ion channels, the variations of the intracellular calcium (Ca(2+)) concentration were explored using a fluorescent marker. The results revealed that ultrasound stimulation induces a rapid hyperpolarization of cell membrane potential when the microbubble is in direct contact with the cell, but the potential returned to its initial value when ultrasound stimulation stopped. The change in cell membrane potential indicates the activation of specific ion channels and depends on the quality of microbubble adhesion to the cell membrane. Microbubbles were shown to induce a mechanical stretch activating BKca channels. Simultaneous Ca(2+) measurements indicate a slow and progressive Ca(2+) increase that is likely a consequence of BKca channels opening not a cause. These results demonstrate that microbubbles' oscillations under ultrasound activation entail modulation of cellular function and signaling by t- riggering the modulation of ionic transports through the cell membrane. Cells response to the mechanical stretch caused by gentle microbubble oscillations is characterized by the opening of BKca stretch channels and a Ca(2+) flux, which might potentially trigger other cellular responses responsible for membrane sonopermeabilization.