No abstract

Staurosporine (STS) and etoposide (Eto) induced apoptosis of the human histiocytic lymphoma cells U937 were studied to determine the role of monovalent ions in apoptotic cell shrinkage. Cell shrinkage, defined as cell dehydration, was assayed by measurement of buoyant density of cells in continuous Percoll gradient. The K⁺ and Na⁺ content in cells of different density fractions was estimated by flame emission analysis. Apoptosis was evaluated by confocal microscopy and flow cytometry of acridine orange stained cells, by flow DNA cytometry and by effector caspase activity. Apoptosis of U937 cells induced by 1 µM STS for 4 h was found to be paralleled by an increase in buoyant density indicating cell shrinkage. An increase in density was accompanied by a decrease in K⁺ content (from 1.1 to 0.78 mmol/g protein), which exceeded the increase in Na⁺ content (from 0.30 to 0.34 mmol/g) and resulted in a significant decrease of the total K⁺ and Na⁺ content (from 1.4 to 1.1 mmol/g). In contrast to STS, 50 µM Eto for 4 h or 0.8-8 µM Eto for 18-24 h induced apoptosis without triggering cell shrinkage. During apoptosis of U937 cells induced by Eto the intracellular K⁺/Na⁺ ratio decreased like in the cells treated with STS, but the total K⁺ and Na⁺ content remained virtually the same due to a decrease in K⁺ content being nearly the same as an increase in Na⁺ content. Apoptotic cell dehydration correlated with the shift of the total cellular K⁺ and Na⁺ content. There was no statistically significant decrease in K⁺ concentration per cell water during apoptosis induced by either Eto (by 13.5%) or STS (by 8%), whereas increase in Na⁺ concentration per cell water was statistically significant (by 27% and 47%, respectively). The data show that apoptosis can occur without cell shrinkage-dehydration, that apoptosis with shrinkage is mostly due to a decrease in cellular K⁺ content, and that this decrease is not accompanied by a significant decrease of K⁺ concentration in cell water.

No abstract

Store-operated Ca 2+ entry (SOCE), a major Ca 2+ signaling mechanism in non-myocyte cells, has recently emerged as a component of Ca 2+ signaling in cardiac myocytes. Though it has been reported to play a role in cardiac arrhythmias and to be upregulated in cardiac disease, little is known about the fundamental properties of cardiac SOCE, its structural underpinnings or effector targets. An even greater question is how SOCE interacts with canonical excitation-contraction coupling (ECC). We undertook a multiscale structural and functional investigation of SOCE in cardiac myocytes from healthy mice (wild type; WT) and from a genetic murine model of arrhythmic disease (catecholaminergic ventricular tachycardia; CPVT). Here we provide the first demonstration of lo cal, transient C a 2+ e ntry (LoCE) events, which comprise cardiac SOCE. Although infrequent in WT myocytes, LoCEs occurred with greater frequency and amplitude in CPVT myocytes. CPVT myocytes also evidenced characteristic arrhythmogenic spontaneous Ca 2+ waves under cholinergic stress, which were effectively prevented by SOCE inhibition. In a surprising finding, we report that both LoCEs and their underlying protein machinery are concentrated at the intercalated disk (ID). Therefore, localization of cardiac SOCE in the ID compartment has important implications for SOCE-mediated signaling, arrhythmogenesis and intercellular mechanical and electrical coupling in health and disease.

We isolated a new proline-rich peptide, ChBac3.4, from leukocytes of the goat (Capra hirca) and determined its amino acid sequence by Edman degradation and mass spectrometry. ChBac3.4 (RFRLPFRRPPIRIHP PPFYPPFRRFL-NH2) had over 50% sequence identity to the Bac5 peptides found in the leukocytes of goats, sheep and cattle.