Neurochemical Research

, Volume 24, Issue 1, pp 69–78 | Cite as

U18666A Inhibits Intracellular Cholesterol Transport and Neurotransmitter Release in Human Neuroblastoma Cells

  • Susan M. Sparrow
  • Jodi M. Carter
  • Neale D. Ridgway
  • Harold W. Cook
  • David M. Byers


To determine if neurochemical function might be impaired in cell models with altered cholesterol balance, we studied the effects of U18666A (3-β-[(2-diethyl-amino)ethoxy]androst-5-en-17-one) on intracellular cholesterol metabolism in three human neuroblastoma cell lines (SK-N-SH, SK-N-MC, and SH-SY5Y). U18666A (≤0.2 μg/ml) completely inhibited low density lipoprotein (LDL)-stimulated cholesterol esterification in SK-N-SH cells, while cholesterol esterification stimulated by 25-hydroxycholesterol or bacterial sphingomyelinase was unaffected or partially inhibited, respectively. U18666A also blocked LDL-stimulated downregulation of LDL receptor and caused lysosomal accumulation of cholesterol as measured by filipin staining. U18666A treatment for 18 h resulted in 70% inhibition of K+-evoked norepinephrine release in phorbol esterdifferentiated SH-SY5Y cells, while release stimulated by the calcium ionophore A23187 was only slightly affected. These results suggest that U18666A may preferentially block a voltage-regulated Ca2+ channel involved in norepinephrine release and that alterations in neurotransmitter secretion might be a feature of disorders such as Niemann-Pick Type C, in which intracellular cholesterol transport and distribution are impaired.

Cholesterol U18666A neuroblastoma SH-SY5Y Niemann-Pick C disease norepinephrine 


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  1. 1.
    Liscum, L. and Underwood, K. W. 1995. Intracellular cholesterol transport and compartmentation. J. Biol. Chem. 270: 15443–15446.Google Scholar
  2. 2.
    Schroeder, F., Frolov, A. A., Murphy, E. J., Atshaves, B. P., Jefferson, J. R., Pu, L. X., Wood, W. G., Foxworth, W. B., and Kier, A. B. 1996. Recent advances in membrane cholesterol domain dynamics and intracellular cholesterol trafficking. Proc. Soc. Exp. Biol. Med. 213:150–177.Google Scholar
  3. 3.
    Lange, Y. and Steck, T. L. 1996. The role of intracellular cholesterol transport in cholesterol homeostasis. Trends Cell Biol. 6:205–208.Google Scholar
  4. 4.
    Morell, P. and Jurevics, H. 1996. Origin of cholesterol in myelin. Neurochem. Res. 21:463–470.Google Scholar
  5. 5.
    Pardridge, W. M. and Mietus, L. J. 1980. Palmitate and cholesterol transport through the blood-brain barrier. J. Neurochem. 34:463–466.Google Scholar
  6. 6.
    Posse de Chaves, E. I., Rusinol, A. E., Vance, D. E., Campenot, R. B., and Vance, J. E. 1997. Role of lipoproteins in the delivery of lipids to axons during axonal regeneration. J. Biol. Chem. 272:30766–30773.Google Scholar
  7. 7.
    Spence, M. W. and Callahan, J. W. 1989. Sphingomyelin-cholesterol lipidoses: the Niemann-Pick group of diseases. Pages 1655–1676, in Scriver, C. R., Baudet, A. L., Sly, W. S., and Valle, D., (eds.) The metabolic basis of inherited disease, McGraw-Hill, New York.Google Scholar
  8. 8.
    Pentchev, P. G., Vanier, M. T., Suzuki, K., and Patterson, M. C. 1995. Niemann-Pick disease type C: a cellular cholesterol lipidosis. Pages 2625–2639, in Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D., (eds.) Metabolic basis of inherited disease, McGraw-Hill, New York.Google Scholar
  9. 9.
    Liscum, L. and Faust, J. R. 1987. Low density lipoprotein (LDL)-mediated suppression of cholesterol synthesis and LDL uptake is defective in Niemann-Pick type C fibroblasts. J. Biol. Chem. 262:17002–17008.Google Scholar
  10. 10.
    Pentchev, P. G., Comly, M. E., Kruth, H. S., Tokoro, T., Butler, J., Sokol, J., Filling-Katz, M., Quirk, J. M., Marshall, D. C., Patel, S., Vanier, M. T., and Brady, R. O. 1987. Group C Niemann-Pick disease: faulty regulation of low density lipoprotein uptake and cholesterol storage in cultured fibroblasts. FASEB J. 1:40–45.Google Scholar
  11. 11.
    Pentchev, P. G., Comly, M. E., Kruth, H. S., Vanier, M. T., Wenger, D. A., Patel, S., and Brady, R. O. 1985. A defect in cholesterol esterification in Niemann-Pick disease (type C) patients. Proc. Natl. Acad. Sci. USA 82:8247–8251.Google Scholar
  12. 12.
    Liscum, L., Ruggiero, R. M., and Faust, J. R. 1989. The intracellular transport of low density lipoprotein-derived cholesterol is defective in Niemann-Pick type C fibroblasts. J. Cell Biol. 108:1625–1636.Google Scholar
  13. 13.
    Carstea, E. D., Morris, J. A., Coleman, K. G., Loftus, S. K., Zhang, D., Cummings, C., Gu, J., Rosenfeld, M. A., Pavan, W. J., Krizman, D. B., Nagle, J., Polymeropoulos, M. H., Sturley, S. L., Ioannou, Y. A., Higgins, M. E., Comly, M., Cooney, A., Brown, A., Kaneski, C. R., Blanchette-Mackie, E. J., Dwyer, N. K., Neufeld, E. B., Chang, T. Y., and Liscum, L. 1997. Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis. Science 277:228–231.Google Scholar
  14. 14.
    Liscum, L. and Faust, J. R. 1989. The intracellular transport of low density lipoprotein-derived cholesterol is inhibited in CHO cells cultured with 3-β-[2-(diethylamino)ethoxy]androst-5-en-17-one. J. Biol. Chem. 264:11796–11806.Google Scholar
  15. 15.
    Butler, J. D., Blanchette-Mackie, J., Goldin, E., O'Neill, R. R., Carstea, G., Roff, C. F., Patterson, M. C., Patel, S., Comly, M. E., Cooney, A., Vanier, M. T., Brady, R. O., and Pentchev, P. G. 1992. Progesterone blocks cholesterol translocation from lysosomes. J. Biol. Chem. 267:23797–23805.Google Scholar
  16. 16.
    Roff, C. F., Goldin, E., Comly, M. E., Cooney, A., Brown, A., Vanier, M. T., Miller, S. P. F., Brady, R. O., and Pentchev, P. G. 1991. Type C Niemann-Pick disease: use of hydrophobic amines to study defective cholesterol transport. Dev. Neurosci. 13:315–319.Google Scholar
  17. 17.
    Rodriguez-Lafrasse, C., Rousson, R., Bonnet, J., Pentchev, P. G., Louisot, P., and Vanier, M. T. 1990. Abnormal cholesterol metabolism in imipramine-treated fibroblast cultures. Similarities with Niemann-Pick type C disease. Biochim. Biophys. Acta 1043:123–128.Google Scholar
  18. 18.
    Phillips, W. A. and Avignan, J. 1963. Inhibition of cholesterol biosynthesis in the rat by 3β-(2-diethylaminoethoxy)androst-5-en-17-one hydrochloride. Proc. Soc. Exp. Biol. 112:233–236.Google Scholar
  19. 19.
    Aikawa, K., Furuchi, T., Fujimoto, Y., Arai, H., and Inoue, K. 1994. Structure-specific inhibition of lysosomal cholesterol transport in macrophages by various steroids. Biochim. Biophys. Acta Lipids Lipid Metab. 1213:127–134.Google Scholar
  20. 20.
    Underwood, K. W., Andemariam, B., McWilliams, G. L., and Liscum, L. 1996. Quantitative analysis of hydrophobic amine inhibition of intracellular cholesterol transport. J. Lipid Res. 37:1556–1568.Google Scholar
  21. 21.
    Pahlman, S., Mamaeva, S., Meyerson, G., Mattsson, M. E. K., Bjelfman, C., Ortoft, E., and Hammerling, U. 1990. Human neuroblastoma cells in culture: a model for neuronal cell differentiation and function. Acta Physiol. Scand. Suppl. 592:25–37.Google Scholar
  22. 22.
    Vaughan, P. F. T., Peers, C., and Walker, J. H. 1995. The use of the human neuroblastoma SH-SY5Y to study the effect of second messengers on noradrenaline release. Gen. Pharmac. 26:1191–1201.Google Scholar
  23. 23.
    Biedler, J. L., Helson, L., and Spengler, B. A. 1973. Morphology and growth, tumorigenicity, and cytogenetics of human neuroblastoma cells in continuous culture. Cancer Res. 33:2643–2652.Google Scholar
  24. 24.
    Goldstein, J. L., Basu, S. K., and Brown, M. S. 1983. Receptor-mediated endocytosis of low-density lipoprotein in cultured cells. Meth. Enzymol. 98:241–260.Google Scholar
  25. 25.
    Sidhu, H. S., Rastogi, S. A. R., Byers, D. M., Cook, H. W., Palmer, F. B. St. C., and Spence, M. W. 1992. Cultured fibroblasts from patients with Niemann-Pick disease type C and type D exhibit distinct defects in cholesterol esterification. Biochim. Biophys. Acta 1124:29–35.Google Scholar
  26. 26.
    Beisiegel, U., Schneider, W. J., Brown, M. S., and Goldstein, J. L. 1982. Immunoblot analysis of low density lipoprotein receptors in fibroblasts from subjects with familial hypercholesterolemia. J. Biol. Chem. 257:13150–13156.Google Scholar
  27. 27.
    Byers, D. M., Douglas, J.-A., Cook, H. W., Palmer, F. B. S. C., and Ridgway, N. D. 1994. Regulation of intracellular cholesterol metabolism is defective in lymphoblasts from Niemann-Pick type C and type D patients. Biochim. Biophys. Acta Mol. Basis Dis. 1226:173–180.Google Scholar
  28. 28.
    Turner, N. A., Rumsby, M. G., Walker, J. H., McMorris, F. A., Ball, S. G., and Vaughan, P. F. T. 1994. A role for protein kinase C subtypes α and ɛ in phorbol-ester-enhanced K+-and carbacholevoked noradrenaline release from the human neuroblastoma SHSY5Y. Biochem. J. 297:407–413.Google Scholar
  29. 29.
    Pahlman, S., Ruusala, A. I., Abrahamsson, L., Odelstad, L., and Nilsson, K. 1983. Kinetics and concentration effects of TPA-induced differentiation of cultured human neuroblastoma cells. Cell Differ. 12:165–170.Google Scholar
  30. 30.
    Pörn, M. I. and Slotte, J. P. 1990. Reversible effects of sphingomyelin degradation on cholesterol distribution and metabolism in fibroblasts and transformed neuroblastoma cells. Biochem. J. 271:121–126.Google Scholar
  31. 31.
    Slotte, J. P. and Bierman, E. L. 1988. Depletion of plasma membrane sphingomyelin rapidly alters the distribution of cholesterol between plasma membranes and intracellular cholesterol pools in cultured fibroblasts. Biochem. J. 250:653–658.Google Scholar
  32. 32.
    Byers, D. M., Morgan, M. W., Cook, H. W., Palmer, F. B. St. C., and Spence, M. W. 1992. Niemann-Pick Type II fibroblasts exhibit impaired cholesterol esterification in response to sphingomyelin hydrolysis. Biochim. Biophys. Acta 1138:20–26.Google Scholar
  33. 33.
    Härmälä, A.-S., Pörn, M. I., Mattjus, P., and Slotte, J. P. 1994. Cholesterol transport from plasma membranes to intracellular membranes is inhibited by 3β-[2-(diethylamino)ethoxy]androst-5-en-17-one. Biochim. Biophys. Acta Lipids Lipid Metab. 1211:317–325.Google Scholar
  34. 34.
    Tabas, I., Zha, X., Beatini, N., Myers, J. N., and Maxfield, F. R. 1994. The actin cytoskeleton is important for the stimulation of cholesterol esterification by atherogenic lipoproteins in macrophages. J. Biol. Chem. 269:22547–22556.Google Scholar
  35. 35.
    Blanchette-Mackie, E. J., Dwyer, N. K., Amende, L. M., Kruth, H. S., Butler, J. D., Sokol, J., Comly, M. E., Vanier, M. T., August, J. T., Brady, R. O., and Pentchev, P. G. 1988. Type-C Niemann-Pick disease: low density lipoprotein uptake is associated with premature cholesterol accumulation in the Golgi complex and excessive cholesterol storage in lysosomes. Proc. Natl. Acad. Sci. USA 85:8022–8026.Google Scholar
  36. 36.
    Turner, N. A., Walker, J. H., Ball, S. G., and Vaughan, P. F. T. 1996. Phorbol ester-enhanced noradrenaline secretion correlates with the presence and activity of protein kinase C-α in human SH-SY5Y neuroblastoma cells. J. Neurochem. 66:2381–2389.Google Scholar
  37. 37.
    Goodall, A. R., Turner, N. A., Walker, J. H., Ball, S. G., and Vaughan, P. F. T. 1997. Activation of protein kinase C-α and translocation of the myristoylated alanine-rich C-kinase substrate correlate with phorbol ester-enhanced noradrenaline release from SH-SY5Y human neuroblastoma cells. J. Neurochem. 68:392–401.Google Scholar
  38. 38.
    Vance, J. E., Pan, D., Campenot, R. B., Bussière, M., and Vance, D. E. 1994. Evidence that the major membrane lipids, except cholesterol, are made in axons of cultured rat sympathetic neurons. J. Neurochem. 62:329–337.Google Scholar
  39. 39.
    Pitas, R. E., Boyles, J. K., Lee, S. H., Hui, D., and Weisgraber, K. H. 1987. Lipoproteins and their receptors in the central nervous system. Characterization of the lipoproteins in cerebrospinal fluid and identification of apolipoprotein B,E (LDL) receptors in the brain. J. Biol. Chem. 262:14352–14360.Google Scholar
  40. 40.
    Ignatius, M. J., Shooter, E. M., Pitas, R. E., and Mahley, R. W. 1987. Lipoprotein uptake by neuronal growth cones in vitro. Science 236:959–962.Google Scholar
  41. 41.
    Lange, Y. and Steck, T. L. 1994. Cholesterol homeostasis. Modulation by amphiphiles. J. Biol. Chem. 269:29371–29374.Google Scholar
  42. 42.
    Sexton, R. C., Panini, S. R., Azran, F., and Rudney, H. 1983. Effects of 3β-[2-(diethylamino)ethoxy]androst-5-ene-17-one on the synthesis of cholesterol and ubiquinone in rat intestinal epithelial cell cultures. Biochemistry 22:5687–5692.Google Scholar
  43. 43.
    Jeng, I., Klemm, N., and Samson, L. 1985. Effect of a hypercholesterolemic agent on cholesteryl ester metabolism in glioblastoma cells. Biochem. Pharmacol. 34:1305–1309.Google Scholar
  44. 44.
    Brown, M. S. and Goldstein, J. L. 1997. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 89:331–340.Google Scholar
  45. 45.
    Casciola, L. A. F., van der Westhuyzen, D. R., Gevers, W., and Coetzee, G. A. 1988. Low density lipoprotein receptor degradation is influenced by a mediator protein(s) with a rapid turnover rate, but is unaffected by receptor up-or down-regulation. J. Lipid Res. 29:1481–1489.Google Scholar
  46. 46.
    Mazzone, T., Basheeruddin, K., Ping, L., Frazer, S., and Getz, G. S. 1989. Mechanism of the growth-related activation of the low density lipoprotein receptor pathway. J. Biol. Chem. 264:1787–1792.Google Scholar
  47. 47.
    Bastiaanse, E. M. L., Höld, K. M., and Van der Laarse, A. 1997. The effect of membrane cholesterol content on ion transport processes in plasma membranes. Cardiovasc. Res. 33:272–283.Google Scholar

Copyright information

© Plenum Publishing Corporation 1999

Authors and Affiliations

  • Susan M. Sparrow
    • 1
  • Jodi M. Carter
    • 1
  • Neale D. Ridgway
    • 1
  • Harold W. Cook
    • 1
  • David M. Byers
    • 1
  1. 1.Atlantic Research Centre, Departments of Pediatrics and BiochemistryDalhousie UniversityHalifaxCanada

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