Temporal evolution of intracellular signaling and gene expression following patterns of membrane depolarization in the pheochromocytoma cell line, PC 12

Abstract

An experimental study was performed to characterize the effects of different patterns of membrane depolarization on undifferentiated pheochromocytoma (PC 12) cells. In response to chronic depolarization, cytoplasmic free Ca2+ ([Ca2+]i) levels increased transiently, then decreased to intermediate levels that were maintained for more than 90 minutes. Short pulses of depolarization, from 1 to 5 minutes in length, also resulted in transient increases in [Ca2+]i. Following a recovery period in a polarized state, a second increase in [Ca2+]i could be induced by repeated depolarization. The activity of signal transduction pathways was also characterized following patterns of depolarization. Chronic depolarization elicited transient (less than 10 minutes in duration) activation of the mitogen-activated protein (MAP) kinases Erkl and Erk2, while the cAMP-response element binding protein (CREB) remained active at intermediate levels for over 60 minutes. Pulsatile depolarization also stimulated Erkl/2 and CREB activation, and the rate of deactivation of the MAP kinases was not found to depend on pulse duration. Lastly, both the MAP kinases and CREB were successfully reactivated by pulsatile stimulation, following recovery periods of greater than 10 minutes in duration. Thus, pulsatile stimulation may be a means of maintaining signaling activity over long periods of time The effect of depolarization on gene expression was determined. Gene expression profiling of PC12 cells over the course of 8 hours following a single or double pulse of stimulation confirmed that a diverse set of genes were regulated by electrical activity. These genes included neural differentiation-specific genes, as well as genes involved in cell cycle control and intracellular signaling.

(cont.) Finally, the gene expression profiles of PC12 cells that were depolarized for one week were also measured. Prolonged depolarization induced a new set of diverse genes, and some of which also have roles in cell cycle control and differentiation. Thus, we conclude from our studies that different patterns of electrical stimulation can have numerous instructive influences on undifferentiated neuron-like cell lines.