Swelling-induced K+ influx in cultured primary astrocytes.

The effect of swelling of cultured primary astrocytes from rat brain in hypotonic medium on K+ influx has been studied. A decrease in osmolality from 310 to 180 mOsm increased the activity of sodium pump (ouabain-inhibited 86Rb+ influx) and Na+,K+,2Cl- cotransport (ouabain-insensitive bumetanide-inhibited 86Rb+ influx) by 70 and 35%, respectively. It is suggested that activation of these transport systems makes it possible to retain a high potassium concentration in the cells under regulatory volume decrease.

Mechanism of activation of Na, K-ATPase in nerve fibres during rhythmic excitation.

The mechanism of activation of Na, K-ATPase in nerve fibres during rhythmic excitation was studied. 3H-ouabain binding to the nerve was found to be dependent on the frequency of rhythmic excitation. During rhythmic excitation 3H-ouabain binding was increased in all nerves tested. The maximum of 3H-ouabain binding in squid and crab nerves was observed at 10 impulses/s, and in frog nerve at 100 impulses/s. The level of bound glycoside decreased during high-frequency excitation. Rhythmic excitation did not change Na, K-ATPase affinity to ouabain, but it appeared to increase the concentration of ouabain sensitive sites in the nerve membrane. The enhancement of 3H-ouabain binding to nerve during rhythmic excitation is interpreted as arising from transformation of "inactive" forms of the enzyme to "active" ones.

Giant glial cell: new insight through mechanism-based modeling.

The paper describes a detailed mechanism-based model of a tripartite synapse consisting of P- and R-neurons together with a giant glial cell in the ganglia of the medical leech (Hirudo medicinalis), which is a useful object for experimental studies in situ. We describe the two main pathways of the glial cell activation: (1) via IP(3) production and Ca(2 +) release from the endoplasmic reticulum and (2) via increase of the extracellular potassium concentration, glia depolarization, and opening of voltage-dependent Ca(2 +) channels. We suggest that the second pathway is the more significant for establishing the positive feedback in glutamate release that is critical for the self-sustained activity of the postsynaptic neuron. This mechanism differs from the mechanisms of the astrocyte-neuron signaling previously reported.

Role of acetylcholine in regulation of interaction between axon and Schwann cell during rhythmic excitation of nerve fibers.

Axon excitation increases the number of acetylcholine receptors (ACR) of the Schwann cell (SC) depending on the frequency of rhythmic excitation (RE) and on intercellular concentrations of K+, Ca2+, and acetylcholine. During RE, activity of axonal acetylcholine esterase is decreased, thus providing for high intercellular acetylcholine concentration. Increased intercellular concentration of acetylcholine activates phosphoinositide-specific phospholipase C (PIPLC) of the myelin nerve fiber. During RE, K+ depolarization and acetylcholine exocytosis can activate Ca2+ entry via Ca2+ channels, thus inducing SC ACR phosphorylation mediated by PIPLC stimulation.

Apoptosis in serum-deprived vascular smooth muscle cells: evidence for cell volume-independent mechanism.

Shrinkage is the earliest hallmark of cells undergoing apoptosis. This study examines the role of this phenomenon in the onset of vascular smooth muscle cell (VSMC) apoptosis triggered by growth factor withdrawal. In hyperosmotic media, VSMC showed the same amplitude of shrinkage but were more resistant to apoptosis than endothelial, epithelial and immune system cells. As with growth factor withdrawal, apoptosis in hyperosmotically-shrunken VSMC was sharply potentiated by transfection with E1A-adenoviral protein and was suppressed by activation of cAMP signaling as well as by the pan-caspase inhibitor z-VAD.fmk. Both cell shrinkage and apoptosis in VSMC-E1A treated with hyperosmotic medium were potentiated under sustained Na+, K+ pump inhibition with ouabain that was in contrast to inhibition of apoptosis documented in ouabain-treated, serum-deprived cells. After 1-hr incubation in serum-deprived medium, VSMC-E1A volume declined by approximately 15%. Transfer from hypotonic to control medium decreased VSMC-E1A volume by approximately 25% without any induction of apoptosis. Neither swelling in hyposmotic medium nor dissipation of the transmembrane gradient of K+ and major organic osmolytes protected serum-deprived VSMC-E1A from apoptosis. Thus, our results show that similarly to immune system, endothelial and epithelial cells, extensive VSMC shrinkage in hyperosmotic medium leads to the development of apoptosis. In contrast to hyperosmotic medium, the modest cell volume decrease occurring in serum-deprived VSMC does not contribute to triggering of the apoptotic machinery.

[3H]-thymidine labelling of DNA triggers apoptosis potentiated by E1A-adenoviral protein.

[(3)H]-thymidine is commonly used to analyze the accumulation of [(3)H]-labeled chromatin fragments in cells undergoing apoptosis. This study shows that [(3)H]-thymidine incorporation within DNA is sufficient per se to inhibit growth and to induce apoptosis in canine kidney epithelial cells and porcine aorta endothelial cells. Despite high-level [(3)H]-thymidine-DNA labeling, rat vascular smooth muscle cells (VSMC) showed only modest inhibition of growth and induction of apoptosis compared to other cell types. Similarly to serum deprivation, apoptosis triggered by [(3)H]-thymidine labeling was sharply potentiated by VSMC transfection with a functional analogue of c-myc, E1A-adenoviral protein (VSMC-E1A), and was suppressed by stimulation of cAMP signaling with forskolin as well as by and Na/K pump inhibition with ouabain. Both apoptosis induction and growth suppression seen in [(3)H]-thymidine-treated VSMC-E1A were reduced by the pan-caspase inhibitor z-VAD.fmk. Thus, our results show that the differential efficiency of the apoptotic machinery determines cell type-specific attenuation of growth in cells with [(3)H]-thymidine-labeled DNA. They also demonstrate that [(3)H]-thymidine-treated and serum-deprived VSMC employ common intermediates of the apoptotic machinery, including steps that are potentiated by E1A-adenoviral protein and inhibited by activation of cAMP signaling as well as by inversion of the intracellular [Na(+)](i)/[K(+)](i) ratio.