Treatment of PC12 cells with nerve growth factor (NGF) produces a rapid and transient increase in calcium uptake into the cells. The increased uptake is maximal after 5 minutes of NGF treatment, but after 15 minutes of NGF treatment, no such increase can be observed. The effect of NGF is partially inhibited by blockers of L-type calcium channels. K-252a, an alkaloid-like kinase inhibitor that usually is found to inhibit the actions of NGF on PC12 cells, produces an increase in calcium uptake similar to, but smaller than, that seen with NGF. NGF had no effect on calcium release under these conditions.

Nerve growth factor stimulates the uptake of radioactive calcium into PC12 cells. This stimulation is inhibited by low concentrations of dideoxyforskolin or staurosporine, and by high concentrations of nifedipine or cadmium. On the other hand, neither dideoxyforskolin nor staurosporine inhibited the stimulation of calcium uptake caused by BK-8644 or adenosine triphosphate (ATP). Nickel inhibited only the effect of ATP on calcium uptake, and actually stimulated the effects of either BK-8644 or nerve growth factor. Down-regulation of L-calcium channels by BK-8644 blocked the subsequent stimulation of calcium uptake by this agent, but not the stimulation by nerve growth factor. Conversely, pre-treatment of the cells with nerve growth factor inhibited the subsequent stimulation of calcium uptake by nerve growth factor, but not the stimulation by BK-8644. The effects of BK-8644 and nerve growth factor on calcium uptake were additive, as were the effects of nerve growth factor and ATP. Phosphatase 2A inhibited the effect of nerve growth factor on calcium uptake, but did not influence the action of BK-8644. On the other hand, calcineurin inhibited the effect of BK-8644 on calcium uptake, but potentiated the action of nerve growth factor. Calmidazolium or fluphenazine also inhibited the effect of nerve growth factor on calcium uptake, but okadaic acid stimulated it. A comparison of the effects of these inhibitors on the actions of various calcium channel agonists shows that the channels on which the action of nerve growth factor is exerted are different than either the L-type calcium channels or the ATP-activated calcium channels.(ABSTRACT TRUNCATED AT 250 WORDS)

PC12 cells are a nerve growth factor-responsive clone derived from a rat pheochromocytoma. The cells contain catecholamines and secrete them in response to depolarizing stimuli and cholinergic agonists. Treatment of the cells with nerve growth factor produces a number of very rapid changes, including the structural rearrangement of the cell membrane, the generation of a number of different second messengers, and the phosphorylation of several proteins. The present studies show that nerve growth factor treatment increases the release of dopamine and norepinephrine from the cells within a few minutes and does so independently of its effect on their metabolism. The experiments indicate that this effect on nerve growth factor is dependent on the presence of extracellular calcium and can be blocked by calcium channel antagonists. K-252a, an alkaloid-like material, usually found to inhibit the actions of nerve growth factor on PC12 cells, also increases the release of catecholamines under these conditions.

1. In pheochromocytoma PC12 cells ATP and, to a lesser extent, 2-methylthioATP stimulate phosphoinositide breakdown, release of intracellular calcium, and influx of external calcium, leading to stimulation of norepinephrine release. In contrast, although UTP also stimulates phosphoinositide breakdown, release of intracellular calcium, and influx of external calcium, there is no stimulation of norepinephrine release. 2. 2-MethylthioATP, presumably acting at P2y receptors, and UTP, presumably acting at P2u receptors, in combination elicit a phosphoinositide breakdown greater than that elicited by either alone. Intracellular levels of calcium measured with Fura-2 increase to greater levels with ATP than with UTP and are sustained, while the UTP intracellular levels of calcium rapidly return to basal values. Both ATP and UTP cause a similar influx of 45 Ca2+ presumably by stimulation of a P2 receptor directly linked to a cation channel. 3. It is proposed that PC12 cells contain two distinct G protein-coupled P2 receptors that activate phospholipase C and a P2 receptor linked to a cation channel. The P2y receptor sensitive to ATP (and to 2-methylthioATP) causes the depletion of a pool of intracellular calcium, sufficient to activate so-called "receptor-operated calcium entry". The sustained elevation of intracellular calcium after ATP treatment is proposed to result in stimulation of norepinephrine release and activation of calcium-dependent potassium channels and sodium-calcium exchange pathways. 4. The P2u receptor sensitive to UTP (and to ATP) causes only a transient elevation in levels of intracellular calcium, perhaps from a different pool, insufficient to activate so-called receptor-operated calcium entry. Further sequelae do not ensue, and the functional role of the UTP-sensitive P2u receptor is unknown.