Short-term exposure to waterborne free silver has acute effects on membrane current of Xenopus oocytes.

Waterborne free silver can cause osmo- and ionoregulatory disturbances in freshwater organisms. The effects of a short-term exposure to extracellular Ag+ ions on membrane currents were investigated in voltage-clamped defolliculated Xenopus oocytes. At a holding potential of -60 mV, ionic silver (1 microM Ag+) increased inward currents (=I(Ag)) from -8+/-2 nA to -665+/-41 nA (n=74; N=27). I(Ag) activated within 2 min of silver exposure and then rose impetuously. This current was largely reversible by washout and repeatable. I(Ag) reversed around -30 mV and rectified slightly at more positive potentials. Na+-free bath conditions reduced the silver-induced current to a smaller but sustained current. The response to silver was abolished by the Cl- channel blockers DIDS and SITS, whereas niflumic acid strongly potentiated I(Ag). Intraoocyte injection of AgNO3 to about 1 mM [Ag]i strongly potentiated I(Ag). Extracellular application of either dithiothreitol (DTT), a compound known to reduce disulfide bridges, or L-cysteine abolished Ag+-activated increase of membrane current. In contrast, n-ethylmaleimide (NEM) which oxidizes SH-groups potentiated I(Ag). Hypoosmotic bath solution significantly increased I(Ag) whereas hyperosmolar conditions attenuated I(Ag). The activation of I(Ag) was largely preserved after chelation of cytosolic Ca2+ ions with BAPTA/AM. Taken together, these data suggest that Xenopus oocytes are sensitive to short-term exposure to waterborne Ag+ ions and that the elicited membrane currents result from extra- and intracellular action of Ag+ ions on peptide moieties at the oocyte membrane but may also affect conductances after internalization.

Mechano-sensitivity of epithelial sodium channels (ENaCs): laminar shear stress increases ion channel open probability.

Epithelial cells are exposed to a variety of mechanical forces, but little is known about the impact of these forces on epithelial ion channels. Here we show that mechanical activation of epithelial sodium channels (ENaCs), which are essential for electrolyte and water balance, occurs via an increased ion channel open probability. ENaC activity of heterologously expressed rat (rENaC) and Xenopus (xENaC) orthologs was measured by whole-cell as well as single-channel recordings. Laminar shear stress (LSS), producing shear forces in physiologically relevant ranges, was used to mechanically stimulate ENaCs and was able to activate ENaC currents in whole-cell recordings. Preceding pharmacological activation of rENaC with Zn2+ and xENaC with gadolinium and glibenclamide largely prevented LSS-activated currents. In contrast, proteolytic cleavage with trypsin potentiated the LSS effect on rENaC whereas the LSS effect on xENaC was reversed (inhibition of xENaC current). Further, we found that exposure of excised outside-out patches to LSS led to an increased ion channel open probability without affecting the number of active channels. We suggest that mechano-sensitivity of ENaC may represent a ubiquitous feature for the physiology of epithelia, providing a putative mechanism for coupling transepithelial Na+ reabsorption to luminal transport.

CFTR-dependent Cl- secretion in Xenopus laevis lung epithelium.

In our present study we used preparations from Xenopus laevis lungs to perform electrophysiological Ussing chamber measurements, unidirectional flux measurements, and employed molecular approaches to elucidate the presence and function of a cystic fibrosis transmembrane conductance regulator (CFTR) homolog in this tissue. Application of different CFTR blockers (NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid), niflumic acid (NFA), glibenclamide, lonidamine, CFTR(inh)-172) to the apical side of the tissues was able to significantly decrease the measured short circuit current (I(SC)) indicating a Cl(-) secretion due to luminal located CFTR channels. This was further supported by a net (36)Cl(-) secretion determined by radioactive tracer flux experiments. Further, Xenopus pulmonary epithelia responded to apical chlorzoxazone exposure - a CFTR activator - and this activated current was inhibited by CFTR(inh)-172. We performed reverse transcription-PCR (RT-PCR) and Western blot analysis and with both approaches we found characteristic signals indicating the presence of a CFTR homolog in Xenopus lung. In addition, we were able to detect CFTR in apical membranes of Xenopus lung slices with immunohistological techniques. We conclude that Xenopus lung epithelium exhibits functional CFTR channels and that this tissue represents a valuable model for the investigation of ion transport properties in pulmonary epithelia.

Organotypic slice culture from human adult ventricular myocardium.

AIMS: Cardiovascular research requires complex and functionally intact experimental models. Due to major differences in the cellular and subcellular composition of the myocardium between species, the use of human heart tissue is highly desirable. To enhance the experimental use of the human myocardium, we established methods for the preparation of vital tissue slices from the adult ventricular myocardium as well as conditions for their long-term preservation in organotypic culture. METHODS AND RESULTS: Human ventricular heart samples were derived from surgical specimens excised during a therapeutic Morrow myectomy and cut into 300 µm thick slices. Slices were either characterized in acute experiments or cultured at a liquid-air interface. Viability and functionality were proven by viability staining, enzyme activity tests, intracellular potential recordings, and force measurements. Precision-cut slices showed high viability throughout 28 days in culture and displayed typical cardiomyocyte action potential characteristics, which enabled pharmacological safety testing on the rapid component of the delayed rectifier potassium current (I(Kr)) and ATP-dependent potassium channels throughout the whole culture period. Constant expression of major ion channels was confirmed by quantitative PCR. Acute slices developed excitation-dependent contractions with a clear preload dependency and a ß-adrenergic response. Contractility and myosin light chain expression decreased during the first days in culture but reached a steady state with reactivity upon ß-adrenergic stimulation being preserved. CONCLUSION: Organotypic heart slices represent a multicellular model of the human myocardium and a novel platform for studies ranging from the investigation of molecular interactions to tissue engineering.

Effect of dronedarone on Na+, Ca2+ and HCN channels.

Previous studies showed that amiodarone causes state-dependent inhibition of Na(+) channels thereby mediating an atrial-selective drug effect. The aim of the present study was to investigate the impact of the new antiarrhythmic compound dronedarone on Na(+), Ca(2+) and hyperpolarization-activated cyclic nucleotide-gated ion channels. Monophasic action potentials (MAP) and effective refractory period (ERP) were studied in arterially perfused left atria and ventricular wedge preparations of the pig. Fast Na(+) and Ca(2+) currents in isolated guinea pig ventricular myocytes as well as human HCN4 channels expressed in Chinese hamster ovary (CHO) cells were investigated with the patch-clamp technique. In left atrial epicardial tissue, dronedarone (3 µM) had no effect on the MAP duration, but the drug caused a significant prolongation of the ERP from 145 ± 9 to 184 ± 17 ms (n = 6; p < 0.05). In guinea pig ventricular myocytes, dronedarone exhibited a state-dependent inhibition of the fast Na(+) channel current with an IC(50) of 0.7 ± 0.1 µM, when the holding potential (V (hold)) was -80 mV. The maximal block at the highest concentration used was 77 ± 8%. In contrast, when V (hold) was -100 mV, inhibition with 10 µM dronedarone was only 9 ± 3% (n = 7). Dronedarone blocked Ca(2+) currents elicited by rectangular pulses at V (hold) = -40 mV with an IC(50) value of 0.4 ± 0.1 µM (maximal block by 10 µM dronedarone, 80 ± 6%), whereas at V (hold) = -80 mV, 10 µM dronedarone blocked only 20 ± 6% (n = 4) of the current. Applying an action potential clamp (V (hold) = -80 mV) yielded an IC(50) of 0.4 ± 0.3 µM. Human HCN4 channels expressed in CHO cells were blocked by dronedarone with an IC(50) of 1.0 ± 0.1 µM. Inhibition of fast Na(+) and Ca(2+) channels by dronedarone depends on the cell's resting membrane potential (state-dependent block) favouring an atrial-selective mode of action. Besides fast Na(+) and Ca(2+) channels, dronedarone also inhibits HCN4 currents. This might contribute to the clinically observed reduction in heart rate seen in patients in sinus rhythm after dronedarone treatment.

Epithelial Na+ channels derived from human lung are activated by shear force.

During breathing the pulmonary epithelial cells are permanently exposed to physical forces and shear force (SF) in particular. In our present study we questioned whether the lung epithelial Na(+) channel (hENaC) responds to shear force. For this purpose ENaC was cloned from human lung tissue, expressed in Xenopus oocytes and functionally characterized by electrophysiological techniques. Shear force in physiological relevant ranges was applied via a fluid stream. By the application of SF we obtained an increased inward current indicating an activation of hENaC. The SF-induced effect was reversible and interestingly, the response to SF was augmented by trypsin due to proteolytic cleavage. The direct activation of hENaC by SF was confirmed in outside-out single channel experiments. In five out of nine recordings an increased NP(O) was observed. From our observations we conclude that lung-derived hENaCs are directly activated by SF and this may represent an important feature for the regulation of pulmonary Na(+) reabsorption and pulmonary fluid homeostasis.

Impact of mechanical stress on ion transport in native lung epithelium (Xenopus laevis): short-term activation of Na+, Cl (-) and K+ channels.

Epithelia, in general, and the lung epithelium, in particular, are exposed to mechanical forces, but little is known about their impact on pulmonary ion transport. In our present study, we employed transepithelial ion transport measurements on Xenopus lung preparations using custom-built Ussing chambers. Tissues were exposed to mechanical stress by increasing the water column (5 cm) at one side of the tissues. Apical exposure to hydrostatic pressure significantly decreased the short circuit current (I (SC): 24 +/- 1%, n = 152), slightly decreased the transepithelial resistance (R (T): 7 +/- 2%, n = 152), but increased the apical membrane capacitance (C (M): 16 +/- 6%, n = 9). The pressure-induced effect was sensitive to Na+ (amiloride), Cl(-) (DIDS, NFA, NPPB) and K+ channel blockers (Ba2+), glibenclamide). Further on, it was accompanied by increased extracellular ATP levels. The results show that mechanical stress leads to an activation of Na+, Cl(-), and K+ conductances in a native pulmonary epithelium resulting in a net decrease of ion absorption. This could be of considerable interest, since an altered ion transport may contribute to pathophysiological conditions, e.g., the formation of pulmonary edema during artificial ventilation.