Epidermal growth factor receptor expression during morphological differentiation of pheochromocytoma cells, induced by nerve growth factor or dibutyryl cyclic AMP

Rat pheochromocytoma cells (clone PC12) possess functional surface receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF). PC12 cells respond to NGF as well as to dibutyryl cyclic AMP (dbcAMP) by arrest of cell proliferation and initiation of morphological differentiation, while EGF acts as a mitogen. Exposure of PC12 cells to NGF for several days resulted in a complete loss of rapid EGF responses, such as membrane ruffling and activation of active K+ transport. EGF binding studies revealed that this loss of EGF responses was due to an almost complete reduction of the number of EGF binding sites. In contrast, exposure of PC12 cells to dbcAMP for 2 days did not affect the rapid EGF responses, despite the morphological differentiation. Moreover, EGF binding studies demonstrated a twofold increase in the number of high-affinity binding sites and a small increase in the number of low-affinity sites. In addition, exposure of the cells to dbcAMP caused a twofold increase of EGF-receptor phosphotyrosine kinase activity. These results indicate that neither EGF-binding or the presence of EGF receptors nor the rapid EGF responses are sufficient for persistent proliferation, on one hand, or sufficient to avoid morphological differentiation, on the other.     

Effects of dibutyryl cyclic AMP and cytochalasin B on cultured human glioma cells.

Cultured human glioma cells (138 MG) exposed to dibutyryl cyclic AMP (dbc-AMP; 0.1--5 mM) attained an arborized shape with thin processes extending from a rounded cell body. Cytochalasin B (CB; 1--1 muM) induced similar morphological changes. The processes in both dbc-AMP and CB treated cells were formed by retraction of the cell margin. Colchicine (1muM) completely and liver treated phalloidin (0.1 mg/ml) partially inhibited the morphological alterations induced by dbc-AMP and CB. Dbc-AMP was found to arrest cell movement, cell division and uptake of 2-deoxy-D-glucose. CB has the same effects but was more potent. The effects of dbc-AMP and CB could be due to interference with a common cellular structure, e.g. microfilaments.     

Bidirectional concentration-dependent effects of glucagon and dibutyryl cyclic AMP on DNA synthesis in cultured adult rat hepatocytes

Glucagon and dibutyryl cyclic AMP exerted both stimulatory and inhibitory effects on hepatocyte DNA synthesis when added to primary monolayer cultures in the presence of serum, dexamethasone, insulin and epidermal growth factor. The stimulation occurred at low concentrations of glucagon (1 pM-1 nM) or dibutyryl cyclic AMP (1 nM-1 microM), while the agents inhibited DNA synthesis at higher concentrations (usually glucagon at over 10 nM or dibutyryl cyclic AMP at over 10 microM). The stimulatory effect was stronger at low cell densities (less than 20 X 10(3) hepatocytes/cm2). When the hepatocytes were cultured at higher densities, stimulatory effects were reduced or absent and the inhibition of (hormone-induced) DNA synthesis by a high concentration of glucagon was much more pronounced than at low cell densities. These results indicate dual, bidirectional, effects of cyclic AMP on hepatocyte DNA synthesis.     

Transformation-dependent effect of dibutyryl cyclic AMP on surface properties of rat cultured cells

The possible differential effect of dibutyryl cyclic AMP on the surface properties of ts-NT3-KR rat cells that express a normal phenotype at 37 degrees C and a transformed morphology and behavior at 33 degrees C has been studied. Electrophoretic examination of glycosylated macromolecules revealed a 350,000 dalton glycoconjugate in phenotypically normal cells but not in the corresponding samples from phenotypically transformed cells or in phenotypically "normal" cells rounded by exposure to the cyclic nucleotide. A decreased exposure of a major 100,000 dalton surface component characteristic of cells that expressed a transformed phenotype, was observed when the corresponding cultures were exposed to dibutyryl cyclic AMP. No change in the 230,000 dalton fibronectinlike molecule of phenotypically normal cells was apparent even in the corresponding cultures exposed to the cyclic nucleotide.     

Effects of dibutyryl cyclic AMP and prostaglandin E1 on cultured human glioma cells

Dibutyryl cyclic AMP (db-cAMP) and prostaglandin E₁ (PGE₁) induced morphological alterations in cultured human glioma cells (138 MG). Cells in serum-free medium, treated with db-cAMP (1 mM) or PGE₁ (10μg/ml), within 1–3 h showed multiple thin processes resembling those of normal glial cells. These processes increased in size during a 24 h incubation. In serum-containing medium the appearance of cells with multiple processes was delayed. The induced morphological alterations were reversible upon exchange with fresh serum-containing but not with serum-deprived medium. Actinomycin D (5 μg/ml) did not prevent the changes induced by PGE₁ or db-cAMP. Inhibition of protein synthesis with cycloheximide (10 μg/ml) did not arrest the initial (1–3 h) changes in morphology but blocked further growth of the processes on prolonged incubation. Vinblastine sulphate (0.1 μg/ml) completely inhibited the alterations induced by PGE₁ or db-cAMP.     

Compartive behavioral effects of dibutyryl cyclic AMP and prostaglandin E1 in mice.

Three behavioral tests, spontaneous locomotor activity (SLMA), exploratory behavior (EB) and rotarod performance (RP), a measure of neuromuscular coordination, were used to stuey the interaction of PGE1 (1 mg/kg i.p., 10 min. pretreatment) with DBcAMP (25 mg/kg i.p., 25 min. pretreatment) in mice. A dose-response relationship of PGE1 (0.01-5.0 mg/kg) to SLMA was determined, with a significant decrease in SLMA produced by a dose of 0.1 mg/kg. decreases in SLMA were produced by PGE1 (79%), DBcAMP (41%) and DBcAMP-PGE1 combination (71%). Similar decreases in EB were observed. Although no significant difference between controls and DBcAMP was observed in RP, 52% of mice tested were RP failures following PGE1 and a 100% failure rate was induced by the combination. Mice were treated with a second injection of DBcAMP or PGE1 or the combination 24 hr following the first injection. Behavioral activity of these mice was observed 25 min (DBcAMP) or 10 min (PGE1) after the second dose was administered. A second injection of DBcAMP failed to decrease SLMA and EB from controls; moreover, SLMA began to return towards control levels as early as 2 hr between injections. The second injection of PGE1 or DBcAMP+PGE1 produced the same behavior as that produced by the first injection. On the basis of these results, the relationship of cyclic nucleotides and PGs to behavioral activity is discussed.     

Inhibitory effects of dibutyryl cyclic AMP and theophylline on the melanosome transformation in the embryonic chick pigmented retina cultured in vitro.

When the retinal pigment epithelial cells of chick embryo are cultured in monolayer conditions, the pigment granules are lost from the cytoplasm. The first structural change in depigmentation is the transformation of pigment granules into the degradative organelles designated as the dense body and melanosome complex. The cells are grown in medium containing DBcAMP of various doses from 10^?5 to 10^?2M. Cell proliferation is retarded by treatment with DBcAMP (10^?3M). The transformation of pigment granules is almost completely prevented in all 1-day cultured cells. In 5-day cultured cells continuously treated with more than 10^?4M, the transformation is not only prevented, but the synthesis of pigment granules is stimulated. A similar result is obtained by the administration of 10^?3M theophylline. 5′-AMP does not prevent the transformation of pigment granules but seems to stimulate the synthesis of pigment granules. On the other hand, cGMP is ineffective both on prevention of transformation and on synthesis of pigment granules. The mechanisms of the transformation of pigment granules are discussed.     

Endogenous cyclic AMP in thyroid cell culture. Effect of thyroid-stimulating hormone and dibutyryl cyclic AMP.

We have studied the variations of endogenous cyclic AMP levels in thyroid cells cultured over a period of 7 days in several conditions: in the presence of thyroid-stimulating hormone or dibutyryl cyclic AMP which both promote the aggregation of isolated cells into follicles, and in their absence when cells develop as a typical monolayer. In follicle-forming cells, the cyclic AMP level was found to rise during the first day of culture, then to fall rapidly. In monolayer-forming cells, the cyclic AMP content slightly increases attaining the same level as found in other cells at the fourth day, which remains stable till the seventh day. We have investigated the response of these cells cultured in the presence of dibutyryl cyclic AMP retain the capability of increasing their cyclic AMP concentration whereas monolayer-forming cells do not preserve this quality of thyroid cells.     

Glucose synthesis by isolated guinea-pig hepatocytes. Effect of cyclic AMP and dibutyryl cyclic AMP.

In isolated guinea-pig hepatocytes, dibutyryl cyclic AMP stimulated gluconeogenesis from 2 mM galactose by 25 and 40% respectively. In the presence of 0.5 mM theophylline, cyclic AMP (0.1 mM) increased glucose synthesis from lactate and galactose by 26 and 34% respectively.     

Differential effects of dibutyryl cyclic AMP on the growth and morphology of an established human lymphocyte line

Summary RPMI 1788 lymphocytes growtn in semi-suspension culture proliferate as separate cells and in clumps. The addition of 10⁻³ m dibutyryl cyclic AMP (Bu₂cAMP) to the culture resulted in the attachment of the cells to the substratum and a subsequent conversion of a portion of the adherent cells to a fibroblast-like morphology. Growth of the adherent cells proceeded at nearly the same rate as that of the control cells. When the cells cultured in the presence of Bu₂cAMP were periodically disturbed, they remained in suspension and under this condition a distinct inhibition of growth by Bu₂cAMP was observed. Cortisol at 10⁻⁵ m, a concentration having no effect on the proliferation of RPMI 1788 cells, when added to cells cultured in the presence of Bu₂cAMP, prevented cell attachment, caused detachment of already adherent cells and thereby brought about the Bu₂cAMP-mediated inhibition of growth in suspension. At a higher concentration (10⁻⁴ m), cortisol alone reduced the growth rate of RPMI 1788 lymphocytes. Under the combined effects of 10⁻⁴ m cortisol and 10⁻³ m Bu₂cAMP, the proliferation and viability of cells in suspension were significantly lower than in the presence of either agent alone.     

Endogenous cyclic AMP in thyroid cell culture. Effect of thyroid-stimulating hormone and dibutyryl cyclic AMP

We have studied the variations of endogenous cyclic AMP levels in thyroid cells cultured over a period of 7 days in several conditions: in the presence of thyroid-stimulating hormone or dibutyryl cyclin AMP which both promote the aggregation of isolated cell into follicles, and in their absence when cells develop as a typical monolayer. In follicle-forming cells, the cyclic AMP level was found to rise during the first day of culture, then to fall rapidly. In monolayer-forming cells, the cyclic AMP content slightly increases attaining the same level as found in other cells at the fourth day, which remains stable till the seventh day. We have investigated the response of these cells to the acute effect of thyroid-stimulating hormone: only cells cultured in the presence of dibutyryl cyclic AMP retain the capability of increasing their cycli AMP concentration whereas monolayer-forming cells do not preserve this quality of thyroid cells.     

Different lipolytic effects of theophylline and dibutyryl cyclic AMP in vivo and in vitro.

Both dcAMP and theophylline are known to promote lipolysis in vitro by increasing intracellular cAMP. Although theophylline stimulates FFA mobilization in vivo as well, a report of low circulating FFA levels in the rat given dcAMP suggested that dcAMP may inhibit lipolysis in the intact animal. To explore this possibility, a comparison of the in vitro and in vivo lipolytic effects of theophylline and dcAMP was made in the young dog. Circulating glycerol and FFA levels rose following the administration of theophylline. While glycerol and FFA fell slightly in puppies given dcAMP, only the FFA change was significant. Epinephrine infusions given alone produced sustained elevations of glycerol and FFA. When theophylline was given in conjunction with ongoing epinephrine infusions, plasma glycerol and FFA levels remained high. On the other hand, epinephrine-stimulated lipolysis was markedly inhibited by dcAMP, as shown by pronounced falls of glycerol and FFA from the elevated levels found with epinephrine alone. In vitro studies involving fragments of puppy adipose tissue reveal that epinephrine, theophylline, and dcAMP promoted glycerol release. In contrast to the in vivo observations, lipolysis was also stimulated by combinations of both epinephrine and theophylline as well as by epinephrine and dcAMP. Thus, theophylline stimulates lipolysis in vitro and in vivo in the puppy. In contrast, dcAMP stimulates lipolysis in vitro but inhibits this action in the intact animal. This important difference in the two pharmacologic agents suggests the need for caution when using them in in vivo studies involving the action of cAMP.     

Effects of cyclic AMP and dibutyryl cyclic AMP on amino acid transport in the isolated rat ovary.

Prepubertal rat ovaries were incubated in medium containing the non-utilizable amino acids alpha-aminoisobutyric acid (AIB-14C) or 1-aminocyclo-pentane-carboxylic acid (cycloleucine-14C). The rate of uptake of the two amino acids was studied in the isolated ovaries after different incubation periods. Addition of 5mM cyclic AMP (cAMP) caused a slight stimulation of the AIB-transport but in higher concentrations (10-25 mM) an inhibition was noted. With dibutyrl cyclic AMP (dbcAMP) a dose-dependent increase was seen with 0.5-5 mM concentrations with no further effect of higher concentrations. Time course studies were performed with both AIB and cycloleucine in presence of 10 mM dbcAMP and increased uptake values were noted at each time studied (30-240 min). The phosphodiesterase inhibitor aminophyline in lower concentrations did not influence AIB-transport but 5-10 mM caused increased uptake values in the ovaries. The stimulatory action of dbcAMP on amino acid transport was augmented by a low concentration of aminophylline (0.5 mM). Experiments were in addition carried out in the presence of puromycin and under these circumstances it was still possible to enhance amino acid transport by addition of dbcAMP. The results are discussed in relation to earlier reported effects of gonadotropins on ovarian amino acid transport.     

Sterol-like compound from sensory ganglia. Effect of a nerve growth factor and insulin on its biosynthesis

1. Sensory ganglia from 8-day-old chick embryos were incubated with a specific nerve growth factor and with insulin. 2. From the total lipid extract of the ganglia a compound with steroid characteristics was isolated. 3. The synthesis of this compound, measured spectrophotometrically, diminished after addition of the nerve growth factor and insulin to the incubation medium. 4. The incorporation of sodium [2-(14)C]acetate and dl-[2-(14)C]mevalonic acid into total lipids of the sensory ganglia was stimulated by the nerve growth factor and insulin, but the radioactivity of the sterol-like compound was slightly lower. The incorporation of labelled mevalonic acid either into total lipids or into the sterol-like compound was about 25% lower. 5. About 20% of the acetate incorporated into total lipids and about 87% of the mevalonic acid were recovered in the sterol-like compound.     

Inhibition by cyclic AMP and dibutyryl cyclic AMP of transport of organic acids in kidney cortex.

1. Cyclic adenosine 3',5'-monophosphate and N-6-2'-O-dibutyryl cyclic adenosine 3',5'-monophosphate decrease the initial entry rate and the steady-state uptake of p-aminohippurate and uric acid by rabbit kidney cortex slices. 2. N-6-2'-O-Dibutyryl adenosine 3'-5'-monophosphate inhibits the tubular transport of p-aminohippurate competitively. 3. Isoproterenol, known to increase cyclic nucleotide concentration of the cortical tubules by activation of adenyl cyclase, decreases p-aminohippurate transport. Antidiuretic hormone which is known to stimulate only medullary adenyl cyclase has no effect on p-amino-hippurate uptake by cortical slices. 4. Theophylline, which inhibits cyclic nucleotide phosphodiesterase and, therefore, enhances the cellular accumulation of endogenous cyclic nucleotide, depresses p-aminohippurate transport.     

Temperature effects on cyclic AMP accumulation in cultured fibroblasts

The kinetic parameters that determine the accumulation of cAMP in WI-38 cells stimulated with prostaglandin E1 have been determined at 37 degrees C and at lower temperatures. For desensitized cells, a reduction of temperatures from 37 degrees to 25 degrees C reduced both rate of synthesis and rate of elimination of cAMP by about 40%. The steady-state accumulation was, therefore, about the same at both temperatures. The extent of desensitization was also shown to be comparable at the two temperatures. It can be inferred that there was appreciable desensitization at 4 degrees C after a period of stimulation of less than one hour. This is contrasted with the behavior of C6-2B glioma cells at the same temperature. Escape of cAMP through the plasma membrane showed a greater temperature dependence than any of the other processes concerned with cAMP accumulation.