Functional expression of dihydropyridine-insensitive calcium channels during PC12 cell differentiation by nerve growth factor (NGF), oncogenic ras,or src tyrosine kinase

1. Recombinant retroviruses were used to introduce a temperature-sensitive v-src gene and oncogenic c-Ha-ras into PC12 cells, and stable cell lines expressing these genes were established. 2. As previously reported, expression of v-src (Alema et al., 1985) or c-Ha-ras (Noda et al., 1985) in PC12 cells results in neurite outgrowth resembling that induced by NGF. We report here that v-src but not oncogenic c-Ha-ras induces a stable morphologic neuronal differentiation similar to treatment with NGF. Oncogenic c-Ha-ras-induced neurite outgrowth is not stable with long-term culture, rather the cells revert to an undifferentiated morphology with altered cell cycle kinetics. 3. The stable neuronal phenotype induced by v-src and NGF is characterized by the functional expression of dihydropyridine-insensitive calcium currents.     

Preparation of affinity-purified,biotinylated tetanus toxin,and characterization and localization of cell surface binding sites on nerve growth factor-treated PC12 cells

Biotinylated derivatives of tetanus toxin were prepared and isolated by chromatofocusing and ganglioside-affinity chromatography. Biotinylation was monitored by the appearance of a 210,00 dalton complex upon SDS-polyacrylamide gel electrophoresis in the presence of avidin, and by selective binding to an avidin-Sepharose gel. At molar biotin:toxin ratios from 11 to 201 only biotinylated derivatives with low toxicity were obtained; these derivatives, however, retained 60–80% of their specific binding affinity for brain synaptosomes. A biotinylated tetanus toxin derivative purified by ganglioside-affinity chromatography was used to identify and localize tetanus toxin binding sites on PC12 cells. Electron microscopic analysis with streptavidin-gold revealed very low levels of tetanus toxin binding sites on the surface of untreated cells, and the appearance of such binding sites during the second week of nerve growth factor-induced differentiation. Examination of micrographs of the differentiated cells indicated that the tetanus toxin binding sites sites are concentrated on the neurites, with relatively few appearing on the cell bodies. Cognate studies using¹²⁵I-labeled, affinity-purified tetanus toxin revealed an increase in PC12 binding capacity from about 0.07 nmol/mg protein in untreated cells to 0.8 nmoles/mg protein in cells treated for 14 days with nerve growth factor. Cells treated in suspension for 2–3 weeks with nerve growth factor do not express tetanus toxin binding sites; upon plating, these cells required one week for the appearance of binding sites, although neurites grew much more rapidly from these primed cells. The high binding capacity of these tetanus toxin sites, as well as their sensitivity to neuraminidase, is indicative of a polysialoganglioside structure. The advantages of biotinylated tetanus toxin derivatives are discussed and the significance of nerve growth factor-differentiated PC12 cells grown as monolayers as a model for the study of the development, localization, and function of neuraminidase-sensitive tetanus toxin binding sites is presented.Abbreviations PBS phosphate-buffered saline - STS sucrose-Tris-serum solution - NGF nerve growth factor - C collagen - PL polylysine - BBG bovine brain ganglioside mixture - GM₁ gafactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GD_1a [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GT_1a [N-aceylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GD_1b galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosyl ceramide - GT_1b [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl-N-acetylneuraminyl] galactosylglucosyl ceramide - NANA N-acetylneuraminic acid     

Properties of the 12-O-Tetradecanoylphorbol-13-Acetate-Stimulated S6 Kinase from Rat Astroglial Cells

The S6 kinase activity of astroglial cells in primary culture stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA) has been studied. This activity was eluted as a single peak at 0.15 M NaCl from a DEAE-Sephacel column. The chromatography of this peak on phosphocellulose revealed an activity eluted at 0.15 M NaCl. This partially purified enzyme had a sedimentation coefficient of 3.7S; Km values were 2 X 10(-5) M for ATP and 10(-6) M for 40S ribosomal subunits. The optimal Mg2+ concentration requirement was 2-3 mM. Mn2+ and Co2+ could substitute for Mg2+ (optimum concentrations 1.5 and 0.8 mM, respectively), but these cations were strong inhibitors in the presence of Mg2+. The enzyme was inhibited by N-ethylmaleimide, indicating that it contained thiol groups. This S6 kinase used ATP, but not GTP, as a phosphate donor, and exhibited great specificity for S6 as phosphate acceptor. Whole histones and protamine were slightly phosphorylated whereas phosvitin, histone H1, and surprisingly the peptide Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala were not phosphorylated. The TPA-stimulated S6 kinase resembles the insulin-, fibroblast growth factor- and cyclic AMP-stimulated enzymes, suggesting that several pathways might activate the same entity.     

Lithium Stimulation of Membrane-Bound Phospholipase C from PC 12 Cells Exposed to Nerve Growth Factor

LiCl stimulated the formation of inositol monophosphate in PC12 cells that had been exposed to nerve growth factor (NGF) for 4-5 days. Half-maximal accumulation was observed at approximately 8 mM LiCl. Stimulation of formation of inositol bisphosphate plus inositol trisphosphate was half-maximal at approximately 1 mM LiCl. With membranes isolated from PC12 cells differentiated with NGF, the hydrolysis of added phosphatidylinositol 4,5-bisphosphate (PIP2) was stimulated by LiCl in a biphasic manner, with the first stimulation half-maximal at approximately 0.7 mM and the second half-maximal at approximately 15 mM LiCl. The apparent Km for PIP2 was lowered in the presence of 1.1 mM LiCl from approximately 200 to approximately 70 microM. Membranes from cells grown in the absence of NGF did not respond to LiCl. Although observations with intact cells are difficult to interpret without ambiguity, the results obtained with isolated membranes support our interpretation of the stimulatory action of lithium in the intact PC12 cells.     

Stimulation of Inositol Incorporation into Lipids of PC 12 Cells by Nerve Growth Factor and Bradykinin

The effects of bradykinin (BK) and lithium on the phosphatidylinositol cycle were examined in PC12 cells cultured for 20 h in the presence [PC12(+)] or in the absence [PC12(-)] of nerve growth factor (NGF). BK (1 microM) induced a small stimulation of the incorporation of myo-[2-3H]inositol into the lipids of PC12(-) cells and a three- to fourfold stimulation of such incorporation into the lipids of PC12 (+) cells. About 15 h of incubation with NGF and greater than 10 min of incubation with BK were needed for maximal stimulation of inositol incorporation by BK. In the presence of 25 mM LiCl, BK stimulated the inositol monophosphate levels nine-fold in PC12 (-) and 30-fold in PC12 (+) cells. After incubation for 20 h with NGF, an increased binding of [3H]BK to the PC12 (+) cells was observed at 4 degrees C. Exposure of the cells for 30 min to 25 mM LiCl enhanced the effect of BK on the inositol incorporation into total inositol lipids, especially in PC12(+) cells. In these cells, LiCl in the presence of BK also increased several-fold the intracellular levels of inositol bisphosphate and inositol trisphosphate.     

Effects of Prolonged Depolarization on the Nicotinic Acetylcholine Receptors of PC12 Cells

To determine whether prolonged depolarization and/or changes in intracellular Ca2+ concentrations stimulate adaptive responses of neuronal nicotinic acetylcholine receptors, PC12 pheochromocytoma cells were grown in medium containing various concentrations of K+. Nicotinic receptor function was determined as carbachol-stimulated uptake of 86Rb+. Cells were exposed to 50 mM K+ for up to 4 days and then allowed to repolarize for 60 min. Under these conditions, no changes in basal or carbachol-stimulated uptake of 86Rb+ were observed. Furthermore, neither the time course of carbachol-stimulated uptake or the carbachol concentration dependence of 86Rb+ uptake was altered. Finally, concurrent depolarization did not affect the functional down-regulation produced by chronic exposure of the cells to carbachol. Thus, neuronal nicotinic acetylcholine receptors on PC12 cells do not appear to be regulated by depolarization or prolonged elevation of the intracellular Ca2+ level.     

Ca2+-activated K+ conductance causes membrane hyperpolarizations in a monkey kidney cell line (JTC-12)

Summary We have previously reported hyperpolarizing membrane potential changes in a monkey kidney cell line (JTC-12) which has characteristics resembling proximal tubular cells. These hyperpolarizations could be observed spontaneously or evoked by mechanically touching adjacent cells. In this report, we have shown further evidence that these hyperpolarizations are elicited by an increase in membrane conductance to K⁺ which is caused by an increase in cytosolic Ca²⁺ concentration. In addition, we have found another type of hyperpolarization which is evoked by applying flow of extracellular fluid to the cell. Intracellular injection of Ca²⁺ and Sr²⁺ evoked hyperpolarizations, while intracellular injection of Mn²⁺ and Ba²⁺ did not. Intracellular injection of EGTA suppressed both spontaneous and mechanically evoked hyperpolarizations. In Ca²⁺-free medium, both spontaneous and flow-evoked hyperpolarizations were not observed, while mechanical stimuli consistently evoked hyperpolarization. In Na⁺-free medium, the incidence of cells showing the spontaneous or flow-evoked hyperpolarization increased, and the amplitude and the duration of the mechanically evoked hyperpolarization became greater. Quinidine inhibited all types of hyperpolarization. These data suggest that hyperpolarizations in JTC-12 cells are due to an increase in Ca²⁺-activated K⁺ conductance.     

Protein Kinase C and Melanocyte Transformation

Melanoma cells which have been isolated from metastatic melanoma tissue are able to survive and proliferate in serum supplemented media. In contrast, normal human melanocytes require the presence of growth stimulators if they are to survive in culture. A tumor promotor, 12–0-tetradecanoyl-phorbol-13-acetate (TPA) and substances that increase intracellular levels of cyclic-adenosine-monophosphate (cAMP), such as cholera toxin or isobutylmethyl xanthine, have been widely used for this purpose. The phorbol diester receptor was shown in 1982 to be the phospholipid- and calcium-dependent enzyme protein kinase C (PKC). We therefore directed our studies to the role of PKC regulation in the growth of normal human melanocytes and their transformation. Our studies show that while melanoma cells are inhibited by TPA, the growth of normal melanocytes is stimulated in a dose-dependent manner. The inhibitor, 1-(5-isoquinolinesulfonyl)-2-methyl-piperizine dihydrochloride (H7), which has been found to be the most specific for PKC, had no effect on the growth of normal melanocytes, but inhibited the growth of melanoma cells in a dose-dependent manner. PKC was isolated from the membrane and cytosol of normal melanocytes and melanoma cells. The basal (resting) levels of PKC activity in normal melanocytes was low compared to that measured in melanoma cells, and after short-term (1 hour) treatment with TPA the PKC activity was greatest at the membrane, with the activity decreasing the cytosol. Upon prolonged (48 hours) treatment with TPA, this redistribution of activity continued in normal melanocytes and the total activity increased. In melanoma cells, however, the total PKC activity decreased, particularly in the membrane fraction. A difference in activity and distribution of the enzyme was also seen after short-term (1 hour) treatment with H7. There was very little effect seen on PKC in normal melanocytes; however, the effect on melanoma cells was similar to that seen after 48 hours of exposure to TPA with a decrease in total activity, particularly in the membrane fraction. These results indicate that the regulation of PKC, in particular its activation by TPA, is altered during the transformation of normal human melanocytes     

TPA对原代白血病细胞的诱导分化作用

本文报告了TPA对32例不同类型白血病细胞的体外分化诱导结果。TPA(1.6×10~-7M)可诱导急性非淋巴细胞(ANLL)白血病细胞迅速出现单核巨噬细胞分化标志:细胞贴壁、胞浆丝状伪足形成,具有类似巨噬细胞的形态改变及相应的细胞化学反应特征。急性淋巴细胞白血病(ALL)和桨细胞白血病(PCL)细胞不发生上述变化,表现为细胞聚集成闭现象。慢性淋巴细胞白血病(CLL)出现桨细胞样形态转化。初发与复发病例的诱导反应相类似。TPA体外诱导分化实验,有助于了解病人白血病细胞的分化潜能,对于鉴别粒单系和淋巴系两类白血病,尤其对于用常规方法分型困难的低分化白血病有一定的临床诊断意义。     

Some metabolic relationships between biopterin and folate: Implications for the “methyl trap hypothesis”

Tetrahydrobiopterin and the folate coenzymes can reciprocally interact in ways that would be useful to the metabolic pathways subserved by both of these coenzymes. Thus, through one of the reactions catalyzed by methylene tetrahydrofolate reductase, 5-CH₃-H₄-folate can regenerate BH₄ from q-BH₂ and q-BH₂ can provide an escape from the methyl trap.Special issue dedicated to Dr. Louis Sokoloff