Sodium channels in cultured neuroblastoma cells grown in high glucose or l-Fucose

Patch clamp techniques were used to record whole cell and single channel Na⁺ currents from NB41A3 neuroblastoma cells grown in culture. Cells were grown for two weeks in control medium or medium supplemented with 30 mm d-glucose of 30 mm l-fucose.Cells exposed to glucose or l-fucose had smaller whole cell Na⁺ currents than cells grown in unsupplemented medium, consistent with earlier studies (Yorek, Stefani & Wachtel, 1994). Whole cell macroscopic currents showed no change in activation or inactivation kinetics. Single channel current properties and opening probability were also unchanged.The number of [³H]saxitoxin binding sites, and therefore the total number of Na⁺ channels, was not reduced in cells grown in glucose or l-fucose (Yorek et al., 1994). Therefore, we conclude that some of the channels must have been rendered nonfunctional by the conditioning media. The finding that single channel properties are not altered suggests that channels become nonfunctional in an all-or-none manner.This work was supported by Merit Review Awards to M.A. Yorek and R.E. Wachtel from the Department of Veterans Affairs and by National Institutes of Health grant DK45453 to M.A. Yorek.     

Peroxynitrite-Induced Cytotoxicity in PC12 Cells: Evidence for an Apoptotic Mechanism Differentially Modulated by Neurotrophic Factors

Abstract: Peroxynitrite is a powerful oxidant formed by the near-diffusion-limited reaction of nitric oxide with superoxide. Large doses of peroxynitrite (>2 m M ) resulted in rapid cell swelling and necrosis of undifferentiated PC12 cells. However, brief exposure to lower concentrations of peroxynitrite (EC₅₀ = 850 µ M ) initially (3–4 h) caused minimal damage to low-density cultures. By 8 h, cytoplasmic shrinkage with nuclear condensation and fragmentation became increasingly evident. After 24 h, 36% of peroxynitrite-treated cells demonstrated these features associated with apoptosis. In addition, 46% of peroxynitrite-treated cells demonstrated DNA fragmentation (by terminal-deoxynucleotide transferase-mediated dUTP-digoxigenin nick end-labeling) after 7 h, which was inhibited by posttreatment with the endonuclease inhibitor aurintricarboxylic acid. Serum starvation also resulted in apoptosis in control cells (23%), the percentage of which was not altered significantly by peroxynitrite treatment. Although peroxynitrite is known to be toxic to cells, the present study provides a first indication that peroxynitrite induces apoptosis. Furthermore, pretreatment of cells with nerve growth factor or insulin, but not epidermal growth factor, was protective against peroxynitrite-induced apoptosis. However, both acidic and basic fibroblast growth factors greatly increased peroxynitrite-initiated apoptosis, to 63 and 70%, respectively. Thus, specific trophic factors demonstrate differential regulation of peroxynitrite-induced apoptosis in vitro.     

Intracellular flux analysis in hybridomas using mass balances and in vitro (13)C nmr

Intracellular fluxes are important in defining cellular physiology and its changes in response to environmental variations. Stoichiometric balances combined with extra cellular metabolite measurements were applied to the estimation of intracellular fluxes and the study of energy metabolism in the hybridoma cell line ATCC CRL 1606. Redundant measurements allowed the evaluation of the consistency of the stoichiometry, measurements, and pseudo-steady-state assumption leading to refinement of the assumed biochemistry and identification of measurement errors. To validate the flux estimates, two batch experiments were performed with glucose labeled in the 1 position with (13)C. The distribution of (13)C in secreted lactate was measured via nuclear magnetic resonance spectroscopy (NMR) and compared to that predicted from the estimated intracellular fluxes. There was good agreement between the measured and estimated isotope distributions, demonstrating the validity of the flux estimates obtained from stoichiometric balances. (c) 1995 John Wiley & Sons, Inc.     

BCL-2-Related Protein Expression in Apoptosis: Oxidative Stress Versus Serum Deprivation in PC12 Cells

Abstract: Expression of the BCL-2 protein family members, BAX, BAK, BAD, BCL-xL, BCL-xS, and BCL-2, was measured (by western blotting using specific antibodies) in PC12 cells before and during apoptosis induced by either H₂O₂ treatment or by serum deprivation and during rescue from apoptosis by nerve growth factor (NGF). H₂O₂-induced apoptosis, as measured by DNA fragmentation, caused: (a) a dose-dependent increase in BAX, (b) a dose-independent increase in BAK, and (c) a dose-dependent inhibition of BAD expression. By comparison, apoptosis induced by serum deprivation resulted in a time-dependent decrease in both BAX and BAK, along with a dramatic and sudden decrease in BAD expression. However, when PC12 cells were incubated in an apoptosis-sparing medium (i.e., NGF-supplemented serum-free medium), both BAX and BAK were increased significantly, whereas BAD expression remained inhibited. BCL-xL expression was increased by H₂O₂ but unaffected by serum deprivation or long-term NGF treatment. Neither BCL-2 nor BCL-xS expression could be detected in PC12 cells under the experimental conditions tested. Our results show that the expression of BAX, BAK, BAD, and BCL-xL is altered in a stimulus-dependent manner but cannot be used to define whether a cell will undergo or survive apoptosis. The similarity between changes in expression of BCL-2-related proteins induced by H₂O₂ exposure and NGF rescue could reflect activation in part of a common antioxidant pathway.     

1α,25-dihydroxyvitamin D3 rapidly alters phospholipid metabolism in the nuclea envelope of osteoblasts

1α,25-Dihydroxyvitamin D₃ (1α, 25-(OH)₂D₃) has been shown to increase cytosolic calcium and inositol trophosphate levels in rat osteosarcoma cells (ROS 17/2.8) and to increase nuclear calcium in these cells. To determine the mechanism(s) of 1α, (OH)₂D₃-induced changes in the calcium, the effect of the hormone on phospholipid metabolism in isolated osteoblast nuclei wa assessed. 1α,25 (OH)₂D₃, 20 nM, increased inositol triphosphate levels in the nuclei after 5 min of treatment. The biologically inactive epimer, 1β,25-(OH)₂D₃, had no significant effect on inositol triphosphate levels. ATP, 1 mM, also increased inositol triphosphate levels in the isolated nuclei after 5 min. 1α,25-(OH)₂D₃, 20 nM, increased calcium in the isolated nuclei in the presence but not in the absence of extranuclear calcium with 5 min. Nuclear calcium was also increased within 5 min by ATP, 1 mM, and inositol triphosphate, 1 mM. The effects of ATP on nuclear calcium was not additive with 1α, 25-(OH)₂D₃, suggesting that these two agents increase nuclear calcium in these osteoblast-like cells by similar mechanisms. In summary, 1α,25-(OH)₂D₃ amd ATP rapidly increase inositol triphosphate levels in isolated from ROS 17/2.8 cells. The hormone, the nucleotide, and the inositol phospholipid nuclear calcium. Thus, the 1α,25-(OH)₂D₃ and ATP effects of nuclear calcium may be mediated by changes in phospholipid metabolism in the nuclei of these osteoblastlike cells. © Wiley-Liss, Inc.     

Tumour-infiltrating lymphocytes mediate lysis of autologous squamous cell carcinomas of the head and neck

Tumour-infiltrating lymphocytes (TIL) and tumours from six patients with squamous cell carcinomas of the head and neck (SCCHN) were investigated. The six tumours all expressed major histocompatibility complex (MHC) class I antigens both in vivo and as tumor cell lines grown in vitro. In addition, the cancer cells either overexpressed the tumour-suppressor gene product p53 or harboured human papilloma virus 16/18 (HPV). The TIL were expanded in vitro in the presence of interleukin-2, immobilised anti-CD3 mAb and soluble anti-CD28 mAb. Expanded TIL cultures contained both CD4+and CD8+T cells, but generally contained few CD56+CD3-cells of the natural killer (NK) phenotype. CD8+T cells dominated the individual TIL cultures from five of the six patients and showed significant autologous tumour cell lysis. In TIL cultures derived from four of these tumour-reactive TIL cultures, killing could be partially blocked by an anti-MHC class I mAb. TIL cultures reacting with autologous tumour cells also showed strong TCR/CD3-redirected cytotoxicity when assayed against hybridoma cells expressing anti-TCR/CD3 mAb as well as natural-killer(NK)-like activity. A number of TIL cultures devoid of autologous tumour cell lysis were capable of lysing the natural-killer(NK)-sensitive K562 cell line suggesting that the SCCHN cells themselves are resistant to NK-like lysis. In conclusion, TIL cultures from head and neck carcinomas contain T cells which, upon expansion in vitro, can lyse autologous tumour cells in a MHC-class-I-restricted fashion. Thus, the results of the present study document that carcinomas of the head and neck in some patients are infiltrated by cytotoxic T cell precursors potentially capable of rejecting the autologous tumour.     

Apoptosis and nutrition: Involvement of amino acid transport system in repression of hybridoma cell death

Mouse hybridoma cells cultured on the verge of starvation-induced apoptosis, i.e. in a medium diluted with saline, proved to serve as a sensitive screening system for apoptosis-suppressing activity of nutrient medium components. Conventional amino acid mixtures were found to suppress the starvation-induced apoptosis, whereas a vitamin mixture was ineffective. (Frank F (1995) Biotechnol. Bioeng. 45: 86–90). Recent experiments showed that suppression of apoptosis, and concurrent resumption of growth, could be achieved by addition of single substances at millimolar concentrations. The set of active substances included certain coded L-amino acids (glycine, alanine, serine, threonine, proline, asparagine, glutamine, histidine), non-coded amino acids (-alanine, taurine, 4-aminobutyric acid), and a non-metabolizable analogue (2-aminoisobutyric acid). This finding shows that some amino acids do not act solely as nutrients, but also as specific signal molecules. The specificity of the effect points to the involvement of adaptively regulated amino acid transport systems A and N in maintaining the balance between triggering and suppression of starvation-induced apoptosis.