[en] Microelectrode arrays (MEAs) recording extracellular field potentials of human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CM) provide a rich data set for functional assessment of drug response. The aim of this work is the development of a method for a systematic analysis of arrhythmia using MEAs, with emphasis on the development of six parameters accounting for different types of cardiomyocyte signal irregularities. We describe a software approach to carry out such analysis automatically including generation of a heat map that enables quick visualization of arrhythmic liability of compounds. We also implemented signal processing techniques for reliable extraction of the repolarization peak for field potential duration (FPD) measurement even from recordings with low signal to noise ratios. We measured hiPS-CM's on a 48 well MEA system with 5 minute recordings at multiple time points (0.5, 1, 2 and 4 h) after drug exposure. We evaluated concentration responses for seven compounds with a combination of hERG, QT and clinical proarrhythmia properties: Verapamil, Ranolazine, Flecainide, Amiodarone, Ouabain, Cisapride, and Terfenadine. The predictive utility of MEA parameters as surrogates of these clinical effects were examined. The beat rate and FPD results exhibited good correlations with previous MEA studies in stem cell derived cardiomyocytes and clinical data. The six-parameter arrhythmia assessment exhibited excellent predictive agreement with the known arrhythmogenic potential of the tested compounds, and holds promise as a new method to predict arrhythmic liability. - Highlights: • Six parameters describing arrhythmia were defined and measured for known compounds. • Software for efficient parameter extraction from large MEA data sets was developed. • The proposed cellular parameter set is predictive of clinical drug proarrhythmia.

No abstract available

No abstract available

[en] The transformed phenotype of rat FE-8 cells transfected by an activated human HRAS gene was suppressed upon fusion with normal cells. An experimental approach was developed to identify and isolate a human gene capable of suppressing the transforming activity of the HRAS oncogene in FE-8 cells. Genomic DNA from human placenta was introduced into FE-8 cells by cotransfection with the plasmid pY3 conferring hygromycin B resistance. Transfectants were selected in medium containing hygromycin B. HRAS-transformed FE-8 cells showed an increased sensitivity toward ouabain when compared to their normal counterparts. Therefore, the population of transfected hygromycin B-resistant cells was treated with ouabain to eliminate cells with a transformed phenotype. Ouabain selection resulted in a small number of cell clones exhibiting a more normal phenotype. The clones had lost the morphology of transformed cells and required anchorage for growth. The tumorigenicity of transfectants in nude mice was reduced by not completely abolished. FE-8 revertants continued to express the p21 RAS protein. Human repetitive sequences contained in the DNA of a secondary transfectant were used for isolation of the suppressor gene from reverted FE-8 cells. The cloned DNA fragment was transfected into tumorigenic FE-8 cells and conferred a partial reversion of the transformed phenotype

[en] The authors investigated whether the LLC-PK₁ epithelial cell lines (which shows many characteristics of proximal tubular cells) also is capable of transporting an organic ion. Suspended LLC-PK₁ cells accumulated tetraethylammonium (TEA). The uptake showed characteristics of a facilitated mechanism; TEA uptake was saturable and temperature-dependent and was inhibited by other organic cations. Quinine and mepiperphenidol were the most potent inhibitors, whereas N¹-methylnicotinamide and morphine inhibited the transport system only slightly at doses of 10^-3 M. Basolateral-to-apical TEA flux through LLC-PK₁ monolayers was five to six times larger than that of mannitol, a nontransported compound, whereas apical-to-basolateral TEA and mannitol fluxes were equal. Only the basolateral-to-apical TEA flux was inhibited by quinine. Under similar experimental conditions, no transport of p-aminohippuric acid was observed. It is concluded that LLC-PK₁ cells are able to transport TEA, as do cells of the proximal tubule

[en] We have previously reported the isolation and partial characterization of DNA repair and/or mutagen-sensitive mutant Chinese hamster cell strains. Here we present the results of a detailed study of the ultraviolet light (UV)-induced mutability of one of these strains, UVs-7, and provide preliminary mutability data on two additional lines, UVr-23 and UVs-40. UVs-7 in extremely deficient in unscheduled DNA synthesis (UDS) but only slightly more sensitive to UV than the parental line. When examined for the UV-inducibility of mutants resistant to ouabain, 6-thioguanine, or diphtheria toxin, UVs-7 was found to be hypermutable at all three loci as compared to the parental line. The degree of hypermutability was not the same for any two loci. UVs-40, a highly UV-sensitive strain, was also found to be hypermutable at the ouabain-resistant (ouar) locus. UVr-23, which is UV-resistant and more proficient at UDS than the parental line, appeared to exhibit a tendency toward hypomutability at both the ouabain(ouar) and 6-thioguanine--resistant (6TGr) loci. Further characterization of all these lines should aid in delineating mammalian mechanisms of DNA repair and mutagenesis

[en] The Na,K-ATPase or Na,K-pump in skeletal muscle is essential for the specific properties of this tissue. Furthermore, it is of importance for Na-K-homeostasis and digitalis tolerance of the organism. Thus, a number of different procedures have been developed for the determination of the concentration of Na,K-pumps in skeletal muscle. The purpose of the present review is to describe and evaluate the methods and results available in the literature as well as in our own studies. Due to the high concentration of unspecific ATP-ases present in crude homogenates purification is usually performed, in general by differential centrifugation. However, as the recovery of the Na,K-ATPase in microsomal fractions is subject to variation and is typically less than a few per cent such preparations are not suitable for quantification of the Na,K-pump. Thus a number of variable or even contradictory results have been obtained. Likewise, the quantification of the Na,K-pump by measurement of ³H-ouabain binding to purified enzyme preparations has been unreliable. Comparative determinations using our different methods showed close agreement under a variety of conditions such as differentiation, K-depletion and hypo- and hyperthyroidism. These conditions were all associated with wide variations in the concentration of Na,K-pumps in skeletal muscles of both laboratory animals and patients. It is concluded that our methods, whether based upon intact muscle cells in vitro or in vivo, muscle biopsies or crude muscle homogenates, offer adequate recovery and reproducibility for the quantitative analysis of the concentration of Na,K-pumps and changes herof in skeletal muscle. (eg)

[en] Elevated plasma levels of an endogenous factor with digoxin-like immunoreactivity (DLIS) was recently found in pregnant women, and it has been postulated to play a role in the regulation of fluids and electrolytes, as well as in the pathogenesis of hypertensive disorders in pregnancy. The authors have studied the plasma levels of DLIS in normal women (before and after treatment with contraceptive pills) and in pregnant women (either normotensive or hypertensive), during the gestional and the post-partum period using a sensitive RIA method. In addition, the authors have measured the inhibition of binding activity of ³H-ouabain to intact erythrocytes in 7 plasma samples collected from healthy adults and in 5 plasma samples of women in the second or third trimester of pregnancy. In 8 normal cycling women DLIS levels were similar during the follicular phase (24.9±6.2 pg/ml d.e.) and the luteal phase (22.6±4.7 pg/ml d.e.9. Six months treatment with different preparations of contraceptive pills did not affect the concentrations of DLIS. In a cross-sectional study performed on 171 healthy pregnant women a significant increase (p<0.001) of DLIS plasma levels was observed throughout pregnancy, while a decrease was found after the delivery. A significantly higher (p<0.01) mean level of ³Houbain extracts of pregnant women as compared to normal adults, with a significant correlation between the data obtained with RIA and RRA method. On the other hand, no significant differences in DLIS levels were found between singleton and 9 twin pregnancies, and also between non-hypertensive and 8 hypertensive pregnant women. This data confirm that the plasma concentration of an endogenous factor (or a group of substances) with cardiac glycoside-like activity is significantly increased in pregnant women. However, further studies are necessary to well charcterize the possible role of DLIS in the pathphysiology of hypertension in pregnancy

[en] The Na⁺/K⁺-ATPase pump was reconstituted in proteoliposomes composed of DPPC:DPPE, which were then spread at the air-water interface using Langmuir technique. In the presence of the enzyme in the liposomes the resulting lipidic layer at the air-water interface became more liquid. The effect of Na⁺/K⁺-ATPase on the morphology of Langmuir lipidic layers was monitored by Brewster angle microscopy, which showed the formation of lattice-like structure in between round-shaped lipid domains. The presence of protein stabilized the DPPC:DPPE mixed monolayer at the air-water interface, which was revealed by surface pressure measurements in time and ascribed to hydrogen bond network formation between the protein and the lipids. The activity of the protein embedded in the lipidic Langmuir layer was measured using spectroscopy and voltammetry. Free phosphate released from ATP reacted with ammonium molybdate and the blue α-Keggin anion formed was detected spectrophotometrically at a wavelength of 710 nm. The results based on spectroscopic assay were complemented with electrochemical methods. The activity of the enzyme could by switched off using the inhibitor – ouabain. The Na⁺/K⁺-ATPase activity (2.62 nmol mg⁻¹ min⁻¹) was similar to the activity of the protein solubilized using detergents (3.21 nmol mg⁻¹ min⁻¹). The slightly lower activity was ascribed to the defined orientation of the embedded protein molecules with respect to the air-water interface needed for its activity at the air–water interface.

[en] Previous studies have shown an increase in ³H-ouabain binding sites or Na,K-pumps in vitro in cultured cells in response to incubation in low K, diuretics or lithium. However, in the present study the administration in vivo of various diuretics or lithium combined with supplementary K was not associated with any significant changes in Na,K-content or ³H-ouabain binding site concentration in rat skeletal muscle. When the diuretics were administered in combination with only the basal K requirement a decrease in both K-content and ³H-ouabain binding site concentration was seen. This indicates that the decrease in ³H-ouabain binding site concentration is not caused by these drugs per se but is secondary to the associated K-depletion. The discrepancy between the results obtained using isolated cells and rat skeletal muscles could be related to the fact that cultured cells are not subjected to the normal growth control of the intact organism. It should be emphasized that results obtained using cultured cells do not necessarily reflect processes taking place in the intact organism. (author)