Many drugs, including some commonly used medications, can cause abnormal heart rhythms and sudden death, as manifest by a prolonged QT interval in the electrocardiogram. Cardiac arrhythmias caused by drug-induced long QT syndrome are thought to result mainly from reductions in the delayed rectifier potassium ion (K+) current IKr. Here, we report a mechanism for drug-induced QT prolongation that involves changes in multiple ion currents caused by a decrease in phosphoinositide 3-kinase (PI3K) signaling. Treatment of canine cardiac myocytes with inhibitors of tyrosine kinases or PI3Ks caused an increase in action potential duration that was reversed by intracellular infusion of phosphatidylinositol 3,4,5-trisphosphate. The inhibitors decreased the delayed rectifier K+ currents IKr and IKs, the L-type calcium ion (Ca2+) current ICa,L, and the peak sodium ion (Na+) current INa and increased the persistent Na+ current INaP. Computer modeling of the canine ventricular action potential showed that the drug-induced change in any one current accounted for less than 50% of the increase in action potential duration. Mouse hearts lacking the PI3K p110α catalytic subunit exhibited a prolonged action potential and QT interval that were at least partly a result of an increase in INaP. These results indicate that down-regulation of PI3K signaling directly or indirectly via tyrosine kinase inhibition prolongs the QT interval by affecting multiple ion channels. This mechanism may explain why some tyrosine kinase inhibitors in clinical use are associated with increased risk of life-threatening arrhythmias.

90024.n B S T R A C T We show how the antibiotic nystatin may be used in conjunction with microelectrodes to resolve transepithelial conductance G t into its components: Ga, apical membrane conductance; Gbl, basolateral membrane conductance; and Gj, junctional conductance. Mucosal addition of nystatin to rabbit urinary bladder in Na+-containing solutions caused Gt to increase severalfold to ca. 460/~mho/p.F, and caused the transepithelial voltage V t to approach +50 mV regardless of its initial value. From measurements of G t and the voltage-divider ratio as a function of time after addition or removal of nystatin, values for Ga, Gbl, and Gj of untreated bladder could be obtained. Nystatin proved to have no direct effect on Gbl or Gj but to increase Ga by about two orders of magnitude, so that the basolateral membrane then provided almost all of the electrical resistance in the transcellular pathway. The nystatin channel in the apical membrane was more permeable to cations than to anions. The dose-response curve for nystatin had a slope of 4.6. Use of nystatin permitted assessment of whether microelectrode impalement introduced a significant shunt conductance into the untreated apical membrane, with the conclusion that such a shunt was negligible in the present experiments. Nystatin caused a hyperpolarization of the basolateral membrane potential in Na+-containing solutions. This may indicate that the Na + pump in this membrane is electrogenic.

Several computational models based on experimental techniques and theories have been proposed to describe cytoskeleton (CSK) mechanics. Tensegrity is a prominent model for force generation, but it cannot predict mechanics of individual CSK components, nor explain the discrepancies from the different single cell stimulating techniques studies combined with cytoskeleton-disruptors. A new numerical concept that defines a multi-structural 3D finite element (FE) model of a single-adherent cell is proposed to investigate the biophysical and biochemical differences of the mechanical role of each cytoskeleton component under loading. The model includes prestressed actin bundles and microtubule within cytoplasm and nucleus surrounded by the actin cortex. We performed numerical simulations of atomic force microscopy (AFM) experiments by subjecting the cell model to compressive loads. The numerical role of the CSK components was corroborated with AFM force measurements on U2OS-osteosarcoma cells and NIH-3T3 fibroblasts exposed to different cytoskeleton-disrupting drugs. Computational simulation showed that actin cortex and microtubules are the major components targeted in resisting compression. This is a new numerical tool that explains the specific role of the cortex and overcomes the difficulty of isolating this component from other networks in vitro. This illustrates that a combination of cytoskeletal structures with their own properties is necessary for a complete description of cellular mechanics.

The repertoire of ionic channels expressed in myocytes freshly isolated from microdissected interlobar and arcuate arteries of rat kidney and their integrative behavior in response to endothelin-1 (ET-1) were studied by identification and characterization of major whole cell current components using patch-clamp technique. In renal microvascular smooth muscle cells (RMSMC) dialyzed with K(+)-containing solution, rapidly inactivating (Ito) and sustained outward K+ currents were identified. Voltage-dependent Ito was categorized as "A" current based on its kinetics, sensitivity to 4-aminopyridine (4-AP), and refractoriness to tetraethylammonium (TEA+). Ca(2+)-activated component of K+ current was completely blocked by 10 mM TEA+, whereas 5 mM 4-AP did not affect this current. Maximal Ca2+ current (ICa) recorded in Cs(+)-loaded RMSMC reached 250 pA when cells were bathed in a solution with 2.5 mM Ca2+. Two patterns of ICa differing in kinetics, voltage range of activation and inactivation, and sensitivity to nifedipine were identified as T and L currents. Ca(2+)-dependent current component showing reversal potential near Cl- current (ECl) and sensitivity to blocking action of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid was identified as Ca(2+)-activated ECl. Activation of RMSMC with ET-1 (1-10 nM) induced elevation of [Ca2+]i and subsequent activation of Ca(2+)-activated ICl, which led to membrane depolarization sufficient to activate voltage-gated Ca2+ channels. ET-1-evoked transient reduction of ICa carried through voltage-gated Ca2+ channels was followed by augmentation of L-type ICa. ET-1-induced mobilization of intracellular Ca2+, accompanied by membrane depolarization, resulted in activation of Ca(2+)-dependent K+ channels, which can play the role of a feedback element terminating ET-1-induced membrane depolarization.