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. 1988 Nov 1;255(3):761–768. doi: 10.1042/bj2550761

Second-messenger control of catecholamine release from PC12 cells. Role of muscarinic receptors and nerve-growth-factor-induced cell differentiation.

J Meldolesi 1, G Gatti 1, A Ambrosini 1, T Pozzan 1, E W Westhead 1
PMCID: PMC1135306  PMID: 2850796

Abstract

The role of various intracellular signals and of their possible interactions in the control of neurotransmitter release was investigated in PC12 cells. To this purpose, agents that affect primarily the cytosolic concentration of Ca2+, [Ca2+]i (ionomycin, high K+), agents that affect cyclic AMP concentrations (forskolin; the adenosine analogue phenylisopropyladenosine; clonidine) and activators of protein kinase C (phorbol esters) were applied alone or in combination to either growing chromaffin-like PC12-cells, or to neuron-like PC12+ cells differentiated by treatment with NGF (nerve growth factor). In addition, the release effects of muscarinic-receptor stimulation (which causes increase in [Ca2+]i, activation of protein kinase C and decrease in cyclic AMP) were investigated. Two techniques were employed to measure catecholamine release: static incubation of [3H]dopamine-loaded cells, and perfusion incubation of unlabelled cells coupled to highly sensitive electrochemical detection of released catecholamines. The results obtained demonstrate that: (1) release from PC12 cells can be elicited by both raising [Ca2+]i and activating protein kinases (protein kinase C and, although to a much smaller extent, cyclic AMP-dependent protein kinase); and (2) these various control pathways interact extensively. Activation of muscarinic receptors by carbachol induced appreciable release responses, which appeared to be due to a synergistic interplay between [Ca2+]i and protein kinase C activation. The muscarinic-induced release responses tended to become inactivated rapidly, possibly by feedback desensitization of the receptor mediated by protein kinase C. Muscarinic inactivation was prevented (or reversed) by agents that increase, and accelerated by agents that decrease, cyclic AMP. Agents that stimulate release primarily through the Ca2+ pathway (ionomycin and high K+) were found to be equipotent in both PC12- and PC12+ cells, whereas the protein kinase C activator 12-O-tetradecanoyl-phorbol 13-acetate was approx. 10-fold less potent in PC12+ cells, when administered either alone or in combination with ionomycin. In contrast, the cell binding of phorbol esters was not greatly modified by NGF treatment. Thus control of neurotransmitter release from PC12 cells is changed by differentiation, with a diminished role of the mechanism mediated by protein kinase C.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baizer L., Weiner N. Regulation of dopamine release from PC12 pheochromocytoma cell cultures during stimulation with elevated potassium or carbachol. J Neurochem. 1985 Feb;44(2):495–501. doi: 10.1111/j.1471-4159.1985.tb05441.x. [DOI] [PubMed] [Google Scholar]
  2. Baker P. F. Cell biology: multiple controls for secretion. Nature. 1984 Aug 23;310(5979):629–630. doi: 10.1038/310629a0. [DOI] [PubMed] [Google Scholar]
  3. Berridge M. J. Inositol trisphosphate and diacylglycerol: two interacting second messengers. Annu Rev Biochem. 1987;56:159–193. doi: 10.1146/annurev.bi.56.070187.001111. [DOI] [PubMed] [Google Scholar]
  4. Brocklehurst K. W., Morita K., Pollard H. B. Characterization of protein kinase C and its role in catecholamine secretion from bovine adrenal-medullary cells. Biochem J. 1985 May 15;228(1):35–42. doi: 10.1042/bj2280035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burgoyne R. D., Norman K. M. Effect of calmidazolium and phorbol ester on catecholamine secretion from adrenal chromaffin cells. Biochim Biophys Acta. 1984 Sep 14;805(1):37–43. doi: 10.1016/0167-4889(84)90034-x. [DOI] [PubMed] [Google Scholar]
  6. Derome G., Tseng R., Mercier P., Lemaire I., Lemaire S. Possible muscarinic regulation of catecholamine secretion mediated by cyclic GMP in isolated bovine adrenal chromaffin cells. Biochem Pharmacol. 1981 Apr 15;30(8):855–860. doi: 10.1016/s0006-2952(81)80007-x. [DOI] [PubMed] [Google Scholar]
  7. Di Virgilio F., Milani D., Leon A., Meldolesi J., Pozzan T. Voltage-dependent activation and inactivation of calcium channels in PC12 cells. Correlation with neurotransmitter release. J Biol Chem. 1987 Jul 5;262(19):9189–9195. [PubMed] [Google Scholar]
  8. Di Virgilio F., Pozzan T., Wollheim C. B., Vicentini L. M., Meldolesi J. Tumor promoter phorbol myristate acetate inhibits Ca2+ influx through voltage-gated Ca2+ channels in two secretory cell lines, PC12 and RINm5F. J Biol Chem. 1986 Jan 5;261(1):32–35. [PubMed] [Google Scholar]
  9. Forsberg E. J., Rojas E., Pollard H. B. Muscarinic receptor enhancement of nicotine-induced catecholamine secretion may be mediated by phosphoinositide metabolism in bovine adrenal chromaffin cells. J Biol Chem. 1986 Apr 15;261(11):4915–4920. [PubMed] [Google Scholar]
  10. Gatti G., Madeddu L., Pandiella A., Pozzan T., Meldolesi J. Second-messenger generation in PC12 cells. Interactions between cyclic AMP and Ca2+ signals. Biochem J. 1988 Nov 1;255(3):753–760. doi: 10.1042/bj2550753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Greene L. A., Rein G. Release, storage and uptake of catecholamines by a clonal cell line of nerve growth factor (NGF) responsive pheo-chromocytoma cells. Brain Res. 1977 Jul 1;129(2):247–263. doi: 10.1016/0006-8993(77)90005-1. [DOI] [PubMed] [Google Scholar]
  12. Greene L. A., Tischler A. S. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2424–2428. doi: 10.1073/pnas.73.7.2424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Harris K. M., Kongsamut S., Miller R. J. Protein kinase C mediated regulation of calcium channels in PC-12 pheochromocytoma cells. Biochem Biophys Res Commun. 1986 Feb 13;134(3):1298–1305. doi: 10.1016/0006-291x(86)90391-8. [DOI] [PubMed] [Google Scholar]
  14. Herrera M., Kao L. S., Curran D. J., Westhead E. W. Flow-injection analysis of catecholamine secretion from bovine adrenal medulla cells on microbeads. Anal Biochem. 1985 Jan;144(1):218–227. doi: 10.1016/0003-2697(85)90109-5. [DOI] [PubMed] [Google Scholar]
  15. Kilpatrick D. L., Ledbetter F. H., Carson K. A., Kirshner A. G., Slepetis R., Kirshner N. Stability of bovine adrenal medulla cells in culture. J Neurochem. 1980 Sep;35(3):679–692. doi: 10.1111/j.1471-4159.1980.tb03707.x. [DOI] [PubMed] [Google Scholar]
  16. Knight D. E., Baker P. F. The phorbol ester TPA increases the affinity of exocytosis for calcium in 'leaky' adrenal medullary cells. FEBS Lett. 1983 Aug 22;160(1-2):98–100. doi: 10.1016/0014-5793(83)80944-2. [DOI] [PubMed] [Google Scholar]
  17. Kongsamut S., Miller R. J. Nerve growth factor modulates the drug sensitivity of neurotransmitter release from PC-12 cells. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2243–2247. doi: 10.1073/pnas.83.7.2243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Malgaroli A., Milani D., Meldolesi J., Pozzan T. Fura-2 measurement of cytosolic free Ca2+ in monolayers and suspensions of various types of animal cells. J Cell Biol. 1987 Nov;105(5):2145–2155. doi: 10.1083/jcb.105.5.2145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Matthies H. J., Palfrey H. C., Hirning L. D., Miller R. J. Down regulation of protein kinase C in neuronal cells: effects on neurotransmitter release. J Neurosci. 1987 Apr;7(4):1198–1206. doi: 10.1523/JNEUROSCI.07-04-01198.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Meldolesi J., Huttner W. B., Tsien R. Y., Pozzan T. Free cytoplasmic Ca2+ and neurotransmitter release: studies on PC12 cells and synaptosomes exposed to alpha-latrotoxin. Proc Natl Acad Sci U S A. 1984 Jan;81(2):620–624. doi: 10.1073/pnas.81.2.620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nishizuka Y. Studies and perspectives of protein kinase C. Science. 1986 Jul 18;233(4761):305–312. doi: 10.1126/science.3014651. [DOI] [PubMed] [Google Scholar]
  22. Ono Y., Kikkawa U., Ogita K., Fujii T., Kurokawa T., Asaoka Y., Sekiguchi K., Ase K., Igarashi K., Nishizuka Y. Expression and properties of two types of protein kinase C: alternative splicing from a single gene. Science. 1987 May 29;236(4805):1116–1120. doi: 10.1126/science.3576226. [DOI] [PubMed] [Google Scholar]
  23. Orellana S. A., Solski P. A., Brown J. H. Phorbol ester inhibits phosphoinositide hydrolysis and calcium mobilization in cultured astrocytoma cells. J Biol Chem. 1985 May 10;260(9):5236–5239. [PubMed] [Google Scholar]
  24. Pocotte S. L., Frye R. A., Senter R. A., TerBush D. R., Lee S. A., Holz R. W. Effects of phorbol ester on catecholamine secretion and protein phosphorylation in adrenal medullary cell cultures. Proc Natl Acad Sci U S A. 1985 Feb;82(3):930–934. doi: 10.1073/pnas.82.3.930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pozzan T., Di Virgilio F., Vicentini L. M., Meldolesi J. Activation of muscarinic receptors in PC12 cells. Stimulation of Ca2+ influx and redistribution. Biochem J. 1986 Mar 15;234(3):547–553. doi: 10.1042/bj2340547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pozzan T., Gatti G., Dozio N., Vicentini L. M., Meldolesi J. Ca2+-dependent and -independent release of neurotransmitters from PC12 cells: a role for protein kinase C activation? J Cell Biol. 1984 Aug;99(2):628–638. doi: 10.1083/jcb.99.2.628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rabe C. S., McGee R., Jr Regulation of depolarization-dependent release of neurotransmitters by adenosine: cyclic AMP-dependent enhancement of release from PC12 cells. J Neurochem. 1983 Dec;41(6):1623–1634. doi: 10.1111/j.1471-4159.1983.tb00873.x. [DOI] [PubMed] [Google Scholar]
  28. Rabe C. S., Schneider J., McGee R., Jr Enhancement of depolarization-dependent neurosecretion from PC12 cells by forskolin-induced elevation of cyclic AMP. J Cyclic Nucleotide Res. 1982;8(6):371–384. [PubMed] [Google Scholar]
  29. Role L. W., Perlman R. L. Both nicotinic and muscarinic receptors mediate catecholamine secretion by isolated guinea-pig chromaffin cells. Neuroscience. 1983 Nov;10(3):979–985. doi: 10.1016/0306-4522(83)90236-1. [DOI] [PubMed] [Google Scholar]
  30. Shoyab M., Todaro G. J. Specific high affinity cell membrane receptors for biologically active phorbol and ingenol esters. Nature. 1980 Dec 4;288(5790):451–455. doi: 10.1038/288451a0. [DOI] [PubMed] [Google Scholar]
  31. Swilem A. M., Hawthorne J. N., Azila N. Catecholamine secretion by perfused bovine adrenal medulla in response to nicotinic activation is inhibited by muscarinic receptors. Biochem Pharmacol. 1983 Dec 15;32(24):3873–3874. doi: 10.1016/0006-2952(83)90166-1. [DOI] [PubMed] [Google Scholar]
  32. Vicentini L. M., Ambrosini A., Di Virgilio F., Meldolesi J., Pozzan T. Activation of muscarinic receptors in PC12 cells. Correlation between cytosolic Ca2+ rise and phosphoinositide hydrolysis. Biochem J. 1986 Mar 15;234(3):555–562. doi: 10.1042/bj2340555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Vicentini L. M., Ambrosini A., Di Virgilio F., Pozzan T., Meldolesi J. Muscarinic receptor-induced phosphoinositide hydrolysis at resting cytosolic Ca2+ concentration in PC12 cells. J Cell Biol. 1985 Apr;100(4):1330–1333. doi: 10.1083/jcb.100.4.1330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Vicentini L. M., Di Virgilio F., Ambrosini A., Pozzan T., Meldolesi J. Tumor promoter phorbol 12-myristate, 13-acetate inhibits phosphoinositide hydrolysis and cytosolic Ca2+ rise induced by the activation of muscarinic receptors in PC12 cells. Biochem Biophys Res Commun. 1985 Feb 28;127(1):310–317. doi: 10.1016/s0006-291x(85)80160-1. [DOI] [PubMed] [Google Scholar]

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