Domains of E1A that bind p105Rb, p130, and p300 are required to block nerve growth factor-induced neurite growth in PC12 cells.

Nerve growth factor (NGF) causes PC12 cells to cease division and undergo sympathetic neuron-like differentiation, including neurite outgrowth. We have tested whether differentiation and division share overlapping control mechanisms in these cells. To do this, we have perturbed the activity of proteins known to participate in cell-cycle regulation by introducing the E1A oncogene or its mutant forms via microinjection into PC12 cells. The E1A protein binds to several putative cell cycle control proteins, including p105Rb (the product of the retinoblastoma susceptibility gene), as well as others of unknown function such as p130, p107, and p300. Similar to previous results, we find that wild-type E1A abrogates NGF-induced neurite extension. However, NGF does cause neurite outgrowth in the presence of E1A mutants known to have greatly reduced binding to either p105Rb and p130 or p300. Our experiments suggest that p105Rb, p130, and p300 may participate either in E1A-mediated inhibition of differentiation or in the NGF signal transduction pathway. We also report here that NGF affects phosphorylation of p105Rb, suggesting that Rb mediates at least some of NGF's effects. Our results raise the possibility that putative cell-cycle control proteins may participate not only in NGF-induced cessation of division but also in differentiation.

Nerve growth factor acts through cAMP-dependent protein kinase to increase the number of sodium channels in PC12 cells.

cAMP-dependent protein kinase (PKA) and phospholipid-dependent protein kinase (PKC) play a role in nerve growth factor (NGF)-mediated differentiation. In PC12 cells, NGF causes neurite outgrowth and increases the number of voltage-gated Na+ channels. Neurite outgrowth involves in part activation of PKC. How NGF regulates Na+ channel number is unknown. Using patch-clamp techniques, we find that agents activating PKC, including phorbol esters and a ras oncogene product (p21) that induces neurites, caused little increase in channel number. In contrast, agents increasing intracellular cAMP were as effective as NGF. A specific protein inhibitor of the PKA catalytic subunit blocked increases by NGF or cAMP. Thus, NGF increases Na+ channel number in PC12 cells in part by activating PKA but apparently not PKC.

Calcium-dependent inactivation of the dihydropyridine-sensitive calcium channels in GH3 cells.

The inactivation of calcium channels in mammalian pituitary tumor cells (GH3) was studied with patch electrodes under voltage clamp in cell-free membrane patches and in dialyzed cells. The calcium current elicited by depolarization from a holding potential of -40 mV passed predominantly through one class of channels previously shown to be modulated by dihydropyridines and cAMP-dependent phosphorylation (Armstrong and Eckert, 1987). When exogenous calcium buffers were omitted from the pipette solution, the macroscopic calcium current through those channels inactivated with a half time of approximately 10 ms to a steady state level 40-75% smaller than the peak. Inactivation was also measured as the reduction in peak current during a test pulse that closely followed a prepulse. Inactivation was largely reduced or eliminated by (a) buffering free calcium in the pipette solution to less than 10(-8) M; (b) replacing extracellular calcium with barium; (c) increasing the prepulse voltage from +10 to +60 mV; or (d) increasing the intracellular concentration of cAMP, either 'directly' with dibutyryl-cAMP or indirectly by activating adenylate cyclase with forskolin or vasoactive intestinal peptide. Thus, inactivation of the dihydropyridine-sensitive calcium channels in GH3 cells only occurs when membrane depolarization leads to calcium ion entry and intracellular accumulation.

Morphological properties and membrane channels of the growth cones induced in PC12 cells by nerve growth factor.

Large growth cones were produced in vitro by nerve growth factor (NGF) treatment of multinucleate cells produced by chemical fusion of cells of the neuron-like clone PC12. These endings were studied both at the light microscopic and ultrastructural levels. The activity of ionic channels at growth cones was recorded with intracellular microelectrodes, patch recording of single channels, and whole cone recording from mechanically isolated growth cones. Morphologically, these large growth cones were characterized by the presence of microspikes and filopodia, by the presence of actin demonstrated immunohistochemically, and by the presence of catecholamine fluorescence. At the ultrastructural level they contained a broad spectrum of organelles with a distribution characteristic of neuronal growth cones, including dense core vesicles, abundant smooth membrane cisternae, microtubules, and a filamentous network. The presence of channels capable of generating action potentials was revealed by intracellular microelectrode recording from the growth cone in the presence of locally applied tetraethylammonium (TEA). TEA appeared to block outward current channels that could effectively shunt inward current activated by depolarization. Action potentials elicited by depolarizing current in the presence of TEA could be blocked reversibly by Cd2+, a specific blocker of Ca channels. These action potentials were often followed by a long after-hyperpolarization lasting hundreds of milliseconds. This after-hyperpolarization was similar to that recorded in the cell body of PC12 cells where it appears to be mediated by Ca-activated K current. Single channel recording from outside-out excised patches of membrane from the growth cones perfused with KF revealed the presence of voltage sensitive Na channels, Ca-activated K channels, and K channels resembling delayed rectifier K channels. Macroscopic currents recorded from mechanically isolated growth cones in the "whole cone" configuration showed rapid inward currents at potentials greater than or equal to -40 mV, followed by delayed outward currents at more positive potentials, a finding providing additional evidence for the presence of Na and K channels in growth cones.

A simple and reliable method for the localization in cell culture of single identifiable cells for ultrastructural analyses.

A simple method for relocating single cells in monolayer cultures for subsequent morphological or ultrastructural analysis is reported. This consists of producing, on the culture dish surface, a nontoxic carbon grid that is preserved during processing for either transmission (TEM) or scanning (SEM) electron microscopy. For TEM studies these grids are readily transferred along with the cells into the embedding plastic, and thus individual grid squares containing a cell(s) of interest can be quickly located, remounted, and sectioned. These grids may be useful for ultrastructural analyses of single cells previously studied electrophysiologically or after microinjection of macromolecules.

Physiological and morphological studies of rat pheochromocytoma cells (PC12) chemically fused and grown in culture.

Cell fusion induced by polyethylene glycol has been used to produce in culture giant multinucleate PC12 cells (up to 300 micron in diameter compared to 10-20 micron for unfused cells). Fused cells, like their unfused counterparts, were found to express various neuronal properties. They contained catecholamines. In the presence of nerve growth factor they extended long processes and expressed Na+, Ca2+, and K+ conductances generally associated with excitable cells. In the absence of nerve growth factor these cells neither grew long processes nor generated Na+-spikes. Other neuronal properties were also observed.

Chemical transmission between rat sympathetic neurons and cardiac myocytes developing in microcultures: evidence for cholinergic, adrenergic, and dual-function neurons.

Electrophysiological studies were made on microcultures (300-500 mum in diameter) in which solitary sympathetic principal neurons from newborn rats grew on previously dissociated rat heart cells. Some neurons inhibited,some excited, and others first inhibited and then excited the cardiac myocytes. Application of drugs provided evidence for secretion of acetylcholine by the first group, catecholamines by the second, and both acetylcholine and catecholamines by the third. Solitary neurons which inhibited themyocytes usually excited themselves at nicotinic synapses (autapses).

Formation of cholinergic synapses between dissociated sympathetic neurons and skeletal myotubes of the rat in cell culture.

Sympathetic principal neurons, dissociated from superior cervical ganglia of newborn rats, were plated into cultures containing rat skeletal myotubes formed from previously plated primary myoblasts. Electrophysiological evidence is presented that the neurons developed cholinergic synapses with the myotubes. In addition, the neurons developed cholinergic synapses with each other as previously reported [O'Lague et al. (1974) Proc. Nat. Acad. Sci. USA 71, 3602-3606]. The acetylcholine receptors of myotubes differed from those of the neurons in their sensitivities to curare and hexamethonium, in a manner expected of adult muscle and ganglionic receptors. alpha-Bungarotoxin blocked synaptic transmission from neuron to myotube, but not from neuron to neuron in the same culture.

Evidence for cholinergic synapses between dissociated rat sympathetic neurons in cell culture.

Sympathetic principal neurons were dissociated from superior cervical ganglia of new-born rats, and grown in cell culture. In electrophysiological experiments two types of excitatory synapses were found. One, which was relatively rare, was shown to operate by electrical transmission. The other, the predominant type, had several characteristics of chemical transmission, and pharmacological evidence indicated it was cholinergic.