Changes in the Distribution of theα3 Na+/K+ATPase Subunit in Heterozygous Lurcher Purkinje Cells as a Genetic Model of Chronic Depolarization during Development

A common assumption of excitotoxic mechanisms in the nervous system is that the ionic imbalance resulting from overstimulation of glutamate receptors and increased Na+and Ca++influx overwhelms cellular energy metabolic systems leading to cell death. The goal of this study was to examine how a chronic Na+channel leak current in developing Purkinje cells in the heterozygous Lurcher mutant (+/Lc) affects the expression and distribution of theα3 subunit of the Na+/K+ATPase pump, a key component of the homeostasis system that maintains ionic equilibrium in neurons. The expression pattern of the catalyticα3 Na+/K+ATPase subunit was analyzed by immunohistochemistry, histochemistry, and Western Blots in wild type (WT) and +/Lccerebella at postnatal days P10, P15, and P25 to determine if there are changes in the distribution of active Na+/K+ATPase subunits in degenerating Purkinje cells. The results suggest that the expression of the catalyticα3 subunit is altered in chronically depolarized +/LcPurkinje cells, although the density of active Na+/K+ATPase pumps is not significantly altered compared with WT in the cerebellar cortex at P15, and then declines from P15 to P25 in the +/Lccerebellum as the +/LcPurkinje cells degenerate.

Temporal Characteristics of Activation, Deactivation, and Restimulation of Signal Transduction following Depolarization in the Pheochromocytoma Cell Line PC12

ABSTRACT This study focuses on the transient and dynamic activation of intracellular signal transduction following different protocols of depolarization. During chronic depolarization, phosphorylation of extracellular signal-regulated kinases (ERKs) was observed to peak and subsequently fall to low levels within 10 min of depolarization. Short periods of depolarization, from 1 to 5 min in duration, also led to phosphorylation of ERK, and the rate of ERK dephosphorylation was not affected by the duration of depolarization. Phosphorylation of the cyclic AMP response element binding protein (CREB) also peaked as a result of chronic depolarization but decreased to intermediate levels that were maintained for more than 1 h. Pulsatile depolarization was explored as a means to circumvent the deactivation of intracellular signaling activity during chronic depolarization. Both ERK and CREB were rephosphorylated by a second period of depolarization that followed a recovery period of 10 min or more. The effects of the durations of depolarization and interpulse recovery on reactivation of ERK and CREB were characterized. Measurements of free cytoplasmic Ca2+ confirmed the transient rise in the intracellular calcium concentration ([Ca2+]i) during chronic depolarization and the pulsatile increase in [Ca2+]i that can be achieved with short periods of depolarization. This study characterizes the dynamic activities of signal transduction following depolarization. Electrical stimulation of neurons induces many cellular changes that unfold over time, and the influx of Ca2+ ions that mediate these events is transient. This study suggests that pulsatile activity may be a means of maintaining signaling activity over long periods of time.