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Biomedical subjects

G Guroff

Publications and source records attributed to G Guroff.

At least 73 records · Page 4Linked to original sources
PubMed1986

Protein kinase C as a component of a nerve growth factor-sensitive phosphorylation system in PC12 cells.

The treatment of PC12 cells with either nerve growth factor or phorbol 12-myristate 13-acetate caused a decrease in the phosphorylation of a soluble 100-kDa protein (Nsp100). After treatment with nerve growth factor, the activity of Ca2+/phospholipid-dependent protein kinase (protein kinase C) in the cytosol was increased. When the cytosol from untreated PC12 cells was preincubated with purified protein kinase C and its cofactors, the phosphorylation of Nsp100 was decreased. The preincubation of cytosol from nerve growth factor-treated PC12 cells with protein kinase C did not decrease Nsp100 phosphorylation further. Moreover, preincubation of partially purified Nsp100 kinase with protein kinase C decreased its ability to phosphorylate Nsp100. These results suggest that the binding of nerve growth factor to its receptor on PC12 cells causes an increase in the activity of protein kinase C in the cytosol and phosphorylation of Nsp100 kinase, which in turn lowers its ability to phosphorylate Nsp100.

Animals↗
PubMed1986

Regulation of nerve growth factor action on Nsp100 phosphorylation in PC12h cells by calcium.

Previous work from these laboratories has shown that in PC12 cells the phosphorylation of a specific soluble protein is decreased by treatment with nerve growth factor. This protein, designated Nsp100, and its kinase have been separated and partially purified from PC12 cells. The present studies have been designed to investigate the role of calcium in this action of nerve growth factor. It is shown here, using PC12h cells, that A23187, a calcium ionophore, and high levels of K+, a depolarizing stimulus, also decrease phosphorylation of Nsp100. Furthermore, the actions of nerve growth factor as well as those of A23187 and high levels of K+ are prevented by treatment of the cells with the calcium chelator EGTA. It is also shown that agents that raise levels of cyclic AMP in the cells, specifically dibutyryl cyclic AMP and cholera toxin, also decrease phosphorylation of Nsp100 but, in addition, increase phosphorylation of tyrosine hydroxylase. The action of these latter agents on Nsp100 is blocked by EGTA, but their action on tyrosine hydroxylase is not, indicating that even agents such as cholera toxin act on Nsp100 through a Ca2+-dependent mechanism.

Animals↗
PubMed1986

Nerve growth factor and gangliosides stimulate the release of glycoproteins from PC12 pheochromocytoma cells.

PC12 pheochromocytoma cells in monolayer cultures secrete increased amounts of glycoproteins into the medium following the addition of nerve growth factor (NGF) or of brain gangliosides. After a 48-h incubation with 50 ng/ml NGF there is approximately a twofold increase in the total [14C]glucosamine-labeled, ethanol-precipitable cellular material released into the medium. Between 30 and 50% of the radioactivity is associated with a glycoprotein (Gpl) of molecular weight of 52,000; the remaining radioactivity is distributed between five and six major bands. Only a small amount (10%) is associated with a glycoprotein of Mr greater than 200,000 which might correspond to the NGF-induced large external glycoprotein. A substantial increase in the release of the glycoproteins is also seen on the addition of a variety of gangliosides including asialo GMl. This increase is independent of the presence of NGF. GMl and GDlb/GTlb but not GDla stimulate release above the levels seen in the presence of NGF. Addition of GDla (2 micrograms/ml) enhances selectively the release of various glycoproteins between 2.6- and 8-fold. The pattern of glycoprotein secretion is similar to that seen with NGF, although Gp2 (Mr 78,000) is more abundant. Stimulation of release by GDla is not accompanied by neurite outgrowth, suggesting that the glycoproteins are not directly associated with neuritogenesis. The release of these glycoproteins following the addition of NGF or gangliosides may relate to the neurotrophic properties that these two entirely different ligands exert on PC12 cells.

Adrenal Gland Neoplasms↗
PubMed1986

A nerve growth factor-sensitive S6 kinase in cell-free extracts from PC12 cells.

Soluble extracts from nerve growth factor (NGF)-stimulated PC12 cells prepared by alkaline lysis show a two- to 10-fold greater ability to phosphorylate the 40S ribosomal protein S6 than do extracts from control cells. The alkaline lysis method yields a preparation of much higher specific activity than does sonication. Half-maximal incorporation of 32P from [32P]ATP into S6 occurred after 4-7 min of NGF treatment. The partially purified NGF-sensitive S6 kinase has a molecular weight of 45,000. It is not inhibited by NaCl, chlorpromazine, or the specific inhibitor of cyclic AMP (cAMP)-dependent protein kinase, nor is it activated by addition of diolein plus phosphatidylserine. Trypsin treatment of either crude extracts or partially purified S6 kinase from control or NGF-treated cells was without effect. These data suggest that the S6 kinase stimulated by NGF is neither cAMP-dependent protein kinase or protein kinase C nor the result of tryptic activation of an inactive proenzyme. Treatment of intact cells with dibutyryl cAMP or 5'-N-ethylcarboxamideadenosine also increases the subsequent cell-free phosphorylation of S6. This observation suggests that cAMP-dependent protein kinase may be involved in the phosphorylation of S6 kinase.

Adrenal Gland Neoplasms↗
PubMed1985

Nerve growth factor-induced increase in the cell-free phosphorylation of a nuclear protein in PC12 cells.

In previous studies from this laboratory (Yu, M.W., Tolson, N. W., and Guroff, G. (1980) J. Biol. Chem. 255, 10481-10492) nerve growth factor treatment of PC12 cells was shown to increase the phosphorylation of a specific nonhistone nuclear protein. In the present work these whole-cell observations have been pursued and a cell-free system developed, based on the detergent treatment devised by Lenk et al. (Lenk, R., Ransom, L., Kaufmann, Y., and Penman, S. (1977) Cell 10, 67-78), in order to explore the nerve growth factor-sensitive phosphorylation system in biochemical detail. Using this preparation it has been shown that treatment of the whole cells with nerve growth factor for 30 min or more leads to a marked increase in the subsequent cell-free phosphorylation of the same nonhistone nuclear protein. A characterization of this phosphorylation indicates that it is quite labile to heat and to structural disruption, that it prefers ATP as phosphate donor, and that it requires Mg2+, but is inhibited by high Mg2+ levels as well as by certain other divalent cations. The site of phosphorylation appears to be on serine residues of the protein, as was the phosphorylation observed previously in whole cells. The use of various inhibitors and stimulators suggests that the kinase catalyzing this phosphorylation is not cAMP-dependent, nor is it similar to protein kinase C or casein kinase. The increased phosphorylation produced by nerve growth factor is not transient, the stimulation being constant for at least 3 days in the continuous presence of nerve growth factor. Increases in the phosphorylation of the same nuclear protein can be seen upon treatment of the cells with other effectors such as epidermal growth factor and dibutyryl cyclic AMP, the latter in spite of the fact that cAMP-dependence could not be established in the cell-free system. Finally, a similar system, with a similar stimulation of phosphorylation due to nerve growth factor treatment, can be prepared from sympathetic ganglia from neonatal animals.

Adenosine Triphosphate↗
PubMed1985

Partial purification and characterization of a nerve growth factor-sensitive kinase and its substrate from PC12 cells.

The cell-free, nerve growth factor-sensitive incorporation of radioactive phosphate into a 100,000-dalton protein (Nsp100), observed in a previous study (End,D., Tolson, N., Hashimoto, S., and Guroff, G. (1983) J. Biol. Chem. 258, 6549-6555), has been characterized and the system fractionated. It is shown here that the decrease in incorporation due to treatment of the cells with nerve growth factor is transient, even in the continued presence of nerve growth factor. The decrease in radioactive phosphate incorporation is due to an inhibition of phosphorylation, not to a stimulation of a dephosphorylation. Evidence is presented to suggest that no soluble cofactors are needed for the phosphorylation and no soluble second messengers are responsible for the inhibition. It is demonstrated that the phosphorylation requires divalent cations; both Mg2+ and Mn2+ are effective in this regard. ATP is the preferred phosphate donor, the phosphorylation is maximal at pH values between 5 and 6, and Na+, K+, and Zn2+ are rather specific inhibitors. The system has been partially purified and the resolved components have been used to show that the kinase and the substrate are separate molecules, that the kinase, not the substrate, is the heat-labile portion, and that the kinase has a molecular weight of 110,000-130,000. Finally, evidence is presented to indicate that the kinase, not the substrate, is the component responsible for the decrease in phosphorylation seen after treatment of the cells with nerve growth factor.

Adrenal Gland Neoplasms↗
PubMed1985

Distribution of Nsp100 and Nsp100 kinase, a nerve growth factor-sensitive phosphorylation system, in rat tissues.

Previous work from this laboratory has shown that in PC12 cells the phosphorylation of a specific soluble protein is decreased by nerve growth factor treatment. The protein, designated Nsp100, and its kinase have been separated and partially purified from PC12 cells. In the present work, the tissue distribution of Nsp100 phosphorylation in 5-day-old and adult rats was studied. In adult rats, phosphorylation of an Nsp100-like protein was observed in brain, adrenal gland, testis, and muscle, but not in liver or kidney. In 5-day-old rats, a similar phosphorylation was observed in brain, adrenal gland, superior cervical ganglia, liver, spleen, kidney, and muscle. In PC12 cells, Nsp100 phosphorylation is completely inhibited by 5 X 10(-5) M Zn2+ and is completely inactivated by treatment at 50 degrees C for 2 min. The phosphorylation of the Nsp100-like protein in both adult and 5-day-old rats showed the same characteristics. Partial purification of Nsp100 and Nsp100 kinase from the brains of 5-day-old rats was carried out using the procedures developed for PC12 cells. Nsp100 and Nsp100 kinase were separated on diethylaminoethyl-Sephacel, and the kinase was eluted with 0.3 M NaCl; the same results have previously been obtained with PC12 cells. Phosphorylated Nsp100 from brain and from PC12 cells was compared by proteolysis on sodium dodecyl sulfate gels; similar peptide patterns were generated from the two samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Gland Neoplasms↗
PubMed1985

The effect of fibroblast growth factor on PC12 cells.

PC12 cells, which differentiate morphologically and biochemically into sympathetic neuron-like cells when treated with nerve growth factor, also respond to fibroblast growth factor. Some of the changes induced by fibroblast growth factor are similar to those seen after nerve growth factor treatment. Specifically, pituitary fibroblast growth factor causes the formation of processes initially comparable to those produced by nerve growth factor. However, in contrast to the outgrowth induced by nerve growth factor, which continues for several days, the outgrowth of processes induced by fibroblast growth factor ceases after about 3 days, even though fresh fibroblast growth factor is added. After about 6 days the processes induced by fibroblast growth factor have virtually disappeared. In this regard the processes induced by fibroblast growth factor are very similar to those induced by dibutyryl cyclic adenosine 3':5'-monophosphate (dBcAMP). The addition of nerve growth factor and fibroblast growth factor together appears to produce a synergistic effect on process formation, as does the simultaneous addition of nerve growth factor and dBcAMP. Cells pretreated (or primed) with nerve growth factor are able to regenerate processes much more rapidly in the presence of nerve growth factor than cells which have not been pretreated. When fibroblast growth factor is added to cells primed with nerve growth factor, more rapid regeneration of processes also occurs. The regeneration of neurites in response to either factor is blocked by the addition of an inhibitor of methylation. The process formation induced by fibroblast growth factor is preceded, as is the outgrowth in response to nerve growth factor treatment, by an induction of ornithine decarboxylase, a decrease in the phosphorylation of a specific cytoplasmic protein, and an increase in the phosphorylation of a specific non-histone nuclear protein. The effects of fibroblast growth factor and of nerve growth factor on ornithine decarboxylase are additive. Fibroblast growth factor does not cause an increase in the activity of acetylcholinesterase; nerve growth factor does. Fibroblast growth factor does not appear to be acting through the nerve growth factor receptor. The binding of iodinated nerve growth factor to PC12 cells is specific and is not inhibited by the presence of fibroblast growth factor. In addition, anti-nerve growth factor serum does not interfere with the action of fibroblast growth factor.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholinesterase↗
PubMed1983

Decreased micrococcal nuclease sensitivity of nuclei from nerve growth factor-treated PC12 cells.

Nuclei from nerve growth factor-treated PC12 cells are more resistant to digestion with micrococcal nuclease than are nuclei from control cells. The production of oligosomal fragments is decreased, as is the generation of Mg2+-soluble products. One interpretation of the data is that differentiation of these cells due to treatment with nerve growth factor involves a decrease in the total number of DNA sequences transcribed.

Animals↗
PubMed1983

Nerve growth factor-induced decrease in the cell-free phosphorylation of a soluble protein in PC12 cells.

Incubation of cell-free extracts from PC12 cells with [32P]ATP leads to the phosphorylation of a 100,000-dalton protein. In extracts from cells treated with nerve growth factor, the labeling of the 100,000-dalton protein is substantially and selectively reduced. Direct quantitation indicates that the reduction is a minimum of 30-50% in the various experiments. The decrease is evident after as little as 15 min of nerve growth factor treatment, and disappears within 2 h after the removal of nerve growth factor. The decrease is dose dependent; a complete response is seen after treatment with 10 ng of nerve growth factor/ml. Some decrease in phosphorylation is also seen after treatment of the cells with epidermal growth factor, 12-O-tetradecanoylphorbol-13-acetate, or 5'-N-ethylcarboxamideadenosine, a potent adenosine receptor agonist, but not after treatment with insulin. The phosphorylation of the 100,000-dalton protein, in extracts from either control or nerve growth factor-treated cells, leads almost exclusively to the formation of phosphothreonine. The addition of equal amounts of extract from untreated cells and extract from nerve growth factor-treated cells produces a level of phosphorylation exactly intermediate between those of the two extracts used separately, indicating the absence of a soluble kinase inhibitor. The data suggest that nerve growth factor treatment produces either a covalent inhibition or a physical removal of the kinase for the 100,000-dalton protein.

Adenosine↗
PubMed1983

Identification of nerve growth factor receptors in primary cultures of chick neural crest cells.

Primary cultures of chick neural crest cells obtained from explanted neural tubes have binding sites for radioiodinated nerve growth factor ([125I]NGF) but not for radioiodinated epidermal growth factor ([125I]EGF). The binding of [125I]NGF was shown to be a specific and saturable process with a high affinity (Kd = 0.3 nM) for the ligand. Despite the expression of these NGF binding sites, incubation of the neural crest cultures with nerve growth factor did not induce neurite outgrowth; no morphological alterations were observed. This was not due to an inability of the cells to express a neuronal phenotype, since the neural crest cells spontaneously differentiated into neurite-bearing cells. However, the nerve growth factor binding sites do appear to be functional receptors, since nerve growth factor could produce a modest induction of ornithine decarboxylase. The quantity of nerve growth factor binding sites seemed to be independent of the phenotype expressed by the neural crest cells, since both pigmented cells and neuron-like neural crest cells exhibited binding. These findings suggest that the differentiation of neural crest cells into mature nerve growth factor-responsive neurons may involve the coupling of nerve growth factor receptors to cellular responses important in the expression of the neuronal phenotype.

Animals↗
PubMed1983

The effects of nerve growth factor on polyamine metabolism in PC12 cells.

Nerve growth factor treatment produces a large increase in the activity of ornithine decarboxylase and a moderate decrease in the activity of S-adenosylmethionine decarboxylase in PC12 cells. These changes are reflected weakly, if at all, in the levels of putrescine, spermidine, and spermine in the cells. The rates of polyamine synthesis are increased somewhat more than the overall levels, but still are not comparable in extent to the increase in the ornithine decarboxylase activity. Inhibitors of ornithine decarboxylase and S-adenosylmethionine decarboxylase have their expected effects on the induction of ornithine decarboxylase and on the activities of both enzymes. Neither inhibitor alone, nor a combination of inhibitors, altered the rate or extent of nerve growth factor-induced neurite outgrowth in the cells.

Adenosine↗
PubMed1982

Effects of 12-0-Tetradecanoylphorbol-13-acetate (TPA) on rat pheochromocytoma (PC12) cells: interactions with epidermal growth factor and nerve growth factor.

The phorbol ester tumor promotor 12-0-tetradecanoylphorbol-13-acetate (TPA) specifically inhibited the binding of radioiodinated epidermal growth factor (125I-EGF) to rat pheochromocytoma (PC12) cells in a noncompetitive fashion with an apparent Ki of 11-26 nM. Both TPA and EGF elicited similar biological responses in PC12 cells including enhanced incorporation of 3H-choline and 32 P-orthophosphate into macromolecules, induction of ornithine decarboxylase, and stimulation of the phosphorylation of a 30,000 MW nonhistone, chromosome-associated protein. These effects were also elicited by nerve growth fact (NGF) which, in contrast to the former agents, is a differentiating stimulus for PC12 cells. The effects of TPA were additive or more than additive to the effects of NGF and EGF. When PC12 cells were induced to differentiate by treatment with NGF for 72 hours, the binding of 125I-EGF and responses to EGF were reduced by approximately 70%. The response of PC12 cells to the tumor promoter TPA was unaffected by treatment with NGF. Thus, the qualitatively similar effects of TPA and EGF seemed to be mediated through separate receptor systems with only the EGF receptor system reduced by NGF treatment.

Animals↗
PubMed1982

Simultaneous visualization of the binding of nerve growth factor and epidermal growth factor to single rat pheochromocytoma (PC12) cells through indirect immunohistofluorescence.

The rat pheochromocytoma cell line, PC12, which has receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF), was used to develop a technique for the simultaneous visualization of separate growth factor receptors by indirect immunohistofluorescence. The cells were incubated with saturating concentrations of nerve growth factor and epidermal growth factor. After fixation, the cells were treated with anti-NGF sheep antiserum and then with antisheep rabbit IgG conjugated with fluorescein; they also were treated with anti-EGF rabbit antiserum and then with anti-rabbit sheep IgG conjugated with rhodamine. Fluorescence microscopy showed that a single PC12 cell bound both NGF and EGF. The fluorescence due to EGF binding was reduced when the cells were grown in the presence of NGF. A similar reduction of fluorescence was observed after addition of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Both manipulations are known to reduce the specific binding of 125I-EGF to these cells. Subclones of PC12 cells, NR11 and NR20, reported not to have NGF receptors, did not demonstrate NGF binding when tested with this indirect immunohistofluorescence method. Thus, the binding of growth factors which is demonstrable by indirect immunohistofluorescence method seems to reflect the presence of the specific cell surface receptors for both peptides on individual PC12 cells.

Adrenal Gland Neoplasms↗
PubMed1981

Nerve growth factor-induced alteration in the response of PC12 pheochromocytoma cells to epidermal growth factor.

PC12 cells, which differentiate morphologically and biochemically into sympathetic neruonlike cells in response to nerve growth fact, also respond to epidermal growth factor. The response to epidermal growth factor is similar in certain respects to the response to nerve growth fact. Both peptides produce rapid increases in cellular adhesion and 2-deoxyglucose uptake and both induce ornithine decarboxylase. But nerve growth factor causes a decreased cell proliferation and a marked hypertrophy of the cells. In contrast, epidermal growth factor enhances cell proliferation and does not cause hypertrophy. Nerve growth factor induces the formation of neuritis; epidermal growth factor does not. When both factors are presented simultaneously, the cells form neurites. Furthermore, the biological response to epidermal growth fact, as exemplified by the induction of ornithine decarboxylase, is attenuated by prior treatment of the cells with nerve growth factor. PC12 cells have epidermal growth factor receptors. The binding of epidermal growth factor to these receptors is rapid and specific, and exhibits an equilibrium constant of 1.9 x 10(-9) M. Approximately 80,000 receptors are present per cell, and this number is independent of cell density. Treatment of the cells with nerve growth factor reduces the amount of epidermal growth factor binding by at least 80 percent. The decrease in receptor binding begins after approximately 12-18 h of nerve growth factor treatment and is complete within 3 d. Scratchard plots indicate that the number of binding sites decreases, not the affinity of the binding sites for epidermal growth factor.

Animals↗