Gordon Guroff

Modulation of retinoid signalling through NGF-induced nuclear export of NGFI-B.

The retinoid–X receptor (RXR) regulates multiple hormonal pathways through heterodimerization with nuclear receptors such as the all-trans retinoic acid receptor (RAR). The orphan nuclear receptor NGFI-B (also called Nur77) can heterodimerize with RXR. Here we show that nerve growth factor (NGF) induces the phosphorylation of Ser 105 of NGFI-B in PC12 phaeochromocytoma cells, resulting in translocation of the NGFI-B–RXR heterodimer complex out of the nucleus using nuclear export signals within NGFI-B. As a consequence of the redistribution of RXR, the transcriptional activity of RXR–RAR is reduced. NGFI-B-mediated nuclear export of receptors may serve as a mechanism for crosstalk between NGF and retinoid pathways.

p-Chlorophenylalanine-Induced Chemical Manifestations of Phenylketonuria in Rats

p-Chlorophenylalanine, a potent inhibitor of phenylalanine hydroxylase in vivo, has been used to simulate phenylketonuria in rats. This inhibitor, when administered with phenylalanine, produces marked elevation of blood and tissue phenylalanine without an increase in tyrosine. Disproportionate ratios of phenylalanine to tyrosine are characteristic of phenylketonuria in humans. The use of p-chlorophenylalanine permits the production of this characteristics amino acid imbalance in experimental animals.

Calcium-Dependent Nerve Growth Factor-Stimulated Hydrolysis of Phosphoinositides in PC12 Cells

The effect of nerve growth factor (NGF) on the hydrolysis of phosphoinositides in PC12 cells was examined. Addition of NGF to PC12 cells prelabeled with [3H]-inositol resulted in an increase in the formation of labeled inositol trisphosphate ([3H]IP3), inositol bisphosphate ([3H]IP2), and inositol monophosphate ([3H]IP), an observation indicating that NGF stimulated hydrolysis of the polyphosphoinositides. The increase in these inositol phosphates was detected as early as 15 s after addition of NGF. In the presence of LiCl, the accumulation of [3H]IP was linear for at least 20 min. The NGF-stimulated accumulation of [3H]IP was dose-dependent with a Kact of 0.17 nM and was dependent on the presence of extracellular calcium. In a calcium-free buffer containing EGTA, the NGF-dependent increase in accumulation of [3H]IP was not seen, and the basal level of [3H]IP accumulation was lower than that observed in the presence of extracellular calcium. Lanthanum inhibited both the basal and NGF-stimulated accumulation of [3H]IP, whereas the calcium ionophore A23187, in the absence of NGF, stimulated an accumulation of [3H]IP. The maximal accumulation of [3H]IP in the presence of A23187 was the same as that observed in the presence of NGF. Incubation of the cells with both A23187 and NGF resulted in an accumulation of [3H]IP that was not significantly different from the effect of either agent alone. These results suggest that NGF rapidly stimulates the hydrolysis of phosphoinositides in PC12 cells and that this NGF-stimulated hydrolysis of phosphoinositides occurs by a calcium-dependent mechanism.

PC12 Cells as a Model of Neuronal Differentiation

In 1975 Tischler and Greene reported the culture of a norepinephrine producing pheochromocytoma previously observed and carried in New England Deaconess rats by Warren and his co-workers (Warren and Chute, 1972; DeLellis et al., 1973). The tumor cells grew readily under standard culture conditions and exhibited a formaldehyde-induced fluorescence characteristic of the presence of catecholamines. Many of the cells produced short processes which were evident within 24 hr of plating. In the presence of nanogram quantities of nerve growth factor (NGF), more processes were evident and within 1 to 2 weeks of plating there were up to 20 times more processes in the presence of NGF.

The Action of Adenosine Analogs on PC12 Cells

Abstract: PC12 cells, a nerve growth factor–responsive clone of rat pheochromocytoma, contain a membrane–bound adenylate cyclase, which can be activated by adenosine analogs. The characteristics of the cyclase response indicate the presence of stimulatory adenosine receptors. Adenosine analogs also produce a marked increase in the ornithine decarboxylase levels of the cells, and the characteristics of this response suggest that it is linked to the adenylate cyclase–stimulatory adenosine receptors. The ornithine decarboxylase response elicited by 5′‐N‐ethyIcarboxamideadenosine (NECA), a potent stimulatory adenosine analog, is synergistic with that produced by nerve growth factor. Differentiation of the cells with nerve growth factor, however, does not substantially alter either the response of cyclase to the adenosine analog or the magnitude of the adenosine–evoked ornithine decarboxylase response. Treatment of the cells with NECA produces an increase in the phosphorylation of a specific non–histone nuclear protein. While causing little or no morphological alteration by itself, NECA is synergistic with nerve growth factor in producing neurite outgrowth in PC12 cells. NECA does not cause an induction of acetylcholinesterase in the cells, nor does it appear to affect the induction of this enzyme by nerve growth factor.

The neurite-promoting effect of fibroblast growth factor on PC12 cells

Treatment of PC12 cells with fibroblast growth factor(s) from either brain or pituitary caused neurite outgrowth comparable to that produced by nerve growth factor. The neurite outgrowth was preceded by a substantial rise in the activity of ornithine decarboxylase.

Nerve growth factor treatment prevents the increase in superoxide produced by epidermal growth factor in PC12 cells.

Stimulation of pheochromocytoma (PC12) cells with the mitogen epidermal growth factor (EGF) produced a rapid and robust accumulation of intracellular reactive oxygen species (ROS), an accumulation which, in other systems, has been shown to be essential for mitogenesis. Brief pretreatment of the cells with nerve growth factor (NGF) suppressed the EGF-mediated ROS increase. EGF failed to produce elevations in ROS in a PC12 variant stably expressing a dominant-negative p21 ras construct (PC12-N17) or in cells pretreated with the MEK inhibitor PD098059. NGF failed to suppress the increase in ROS in the PC12 variant nnr5, which lacks p140 trk receptors. The suppression of the increase in ROS by NGF was restored in nnr5 cells stably expressing p140 trk (nnr5-trk), but NGF failed to prevent the increase in ROS in nnr cells expressing mutant p140 trk receptors that lack binding sites for Shc and phospholipase Cγ. Among several inhibitors of superoxide-generating enzymes, only the lipoxygenase inhibitor, nordihydroguaiaretic acid reduced EGF-mediated ROS accumulation. The inhibitory action of NGF on ROS production was mimicked by the nitric oxide donor, sodium nitroprusside, and was blocked by an inhibitor of nitric-oxide synthetase, l-nitroarginine methyl ester. These results suggest a novel mechanism for the rapid interruption of mitogenic signaling by the neurotrophin NGF.

Nerve growth factor-induced reduction in epidermal growth factor responsiveness and epidermal growth factor receptors in PC12 cells: An aspect of cell differentiation

Abstract The rat pheochromocytoma clone PC12 responds to nerve growth factor through the expression of a number of differentiated neuronal properties. One of the most rapid changes is a large, transient increase in the activity of ornithine decarboxylase. These cells also show an increase in ornithine decarboxylase activity in response to the mitogen, epidermal growth factor, but do not respond morphologically as they do to nerve growth factor. Specific, high-affinity epidermal growth factor receptors are present on the cells. When the cells are differentiated with nerve growth factor, the response to epidermal growth factor is markedly diminished and there is a marked reduction in the binding of epidermal growth factor to the cells.

A simple radioisotope assay for phenylalanine hydroxylase

Abstract An isotopic assay for phenylalanine hydroxylase activity has been devised. The assay involves incubation of the enzyme with p -tritio- l -phenylalanine followed by the addition of N -iodosuccinimide to iodinate the tyrosine produced. The tritium released by this combination appears as tritiated water and is proportional to the amount of phenylalanine hydroxylated. Among the advantages of this method are its speed, simplicity, applicability to crude preparations, and utility in studies of the effects of p -phenols on hydroxylating systems. It should also prove valuable in the study of clinical or experimental phenylketonuria.

Hydroxylation of alkyl and halogen substituted anilines and acetanilides by microsomal hydroxylases

Abstract The hydroxylation of a variety of halo and alkyl substituted anilines and acetanilides with hepatic microsomal preparations was studied. 4 Chloro- and 4-fluoro-acetanilides and anilines were hydroxylated to the corresponding 4-hydroxy derivatives with loss of halogen. The 4-chloro derivatives were extremely poor substrates for the aryl hydroxylases. 3-Methyl-, 3-chloro-, and 3-fluoro-acetanilides were metabolized to the corresponding 4-hydroxy-3-substituted acetanilide. 4-Methylaniline, 4-methyl-acetanilide, and 4-ethylacetanilide were not substrates for aryl hydroxylation, but instead underwent side-chain oxidation to form, respectively, 4-hydroxymethylaniline, 4-hydroxymethylacetanilide, and 4-(1′-hydroxyethyl)acetanilide. The hydroxymethyl compounds were further oxidized to aldehydes.