In Vitro Cellular & Developmental Biology

, Volume 26, Issue 4, pp 335–347 | Cite as

Monolayer co-culture of rat heart cells and bovine adrenal chromaffin paraneurons

  • J. M. Trifaró
  • R. Tang
  • M. L. Novas
Regular Papers
Received:
Accepted:

Summary

This paper describes a method for the preparation of co-cultures of rat heart cells and bovine adrenal chromaffin paraneurons. The most suitable condition for heart cell isolation was when a combination of paraneurons. The most suitable condition for heart cell isolation was when a combination of trypsin-DNAse I in Locke's solution was used for digestion. The best co-culture conditions were obtained when 106 heart cells were plated on 7- to 8-d-old adrenal chromaffin paraneuron cultures containing 0.5×106 cells per 35-mm diameter culture dishes. Measurements of DNA (heart cells and chromaffin paraneurons), monitoring of beating frequency (heart cells), and catecholamine (chromaffin paraneurons) levels and release indicated that both cell types remain viable and functional, for several weeks. Heart cells started their characteristic contractile activity 24 h earlier when plated either on viable or lysed chromaffin paraneurons, an effect apparently due to faster surface adhesion of heart cells. The beating frequency of heart cells increased after treatment of co-cultures with either noradrenaline or nicotine, with the latter agent acting indirectly through, the release of chromaffin paraneuron catecholamines. Propranolol produced a dose-related inhibition of the responses to either noradrenaline or nicotine, thus suggesting that the increase in myocyte's beating activity was mediated through β-receptors. Anti-myosin and anti-dopamine-β-hydroxylase immunostaining was used for cell type identification and for the demonstration of body-to-body and process-to-process contacts between adrenal chromaffin paraneurons and heart cells. This co-culture system will serve as a starting point of further studies directed to understand a) the influence of a cell type on the development and on the phenotypic characteristics of a second cell type and b) the interaction of cells derived from different organs and species.

Key words

chromaffin paraneuron co-culture dopamine β-hydroxylase immunohistochemistry myocytes myosin 
This is a preview of subscription content, log in to check access.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Anton, A. H.; Sayre, D. F. A study of the factors affecting the aluminum oxide trihydroxyindole procedure for the analysis of catecholamines. J. Pharmacol. Exp. Ther. 138: 360–371; 1962.PubMedGoogle Scholar
  2. 2.
    Bader, M. F.; Ciesielski-Treska, J.; Thierse, D., et al. Immunocytochemical study of microtubules in chromaffin cells in culture and evidence that tubulin is not an integral protein of the chromaffin granule membrane. J. Neurochem. 37: 917–933; 1981.PubMedCrossRefGoogle Scholar
  3. 3.
    Bader, M. F.; Georges, E.; Mushynski, W. E., et al. Neurofilament proteins in cultured chromaffin cells. J. Neurochem. 43: 1180–1193; 1984.PubMedCrossRefGoogle Scholar
  4. 4.
    Boder, G. B.; Johnson, I. S. Comparative effects of some cardioactive agents on automaticity of cultured heart cells. J. Mol. Cell. Cardiol. 4: 453–463; 1972.PubMedCrossRefGoogle Scholar
  5. 5.
    Boksa, P. Acetylcholine synthesis by adult bovine adrenal chromaffin cell culture. J. Neurochem. 45: 1254–1261; 1985.PubMedCrossRefGoogle Scholar
  6. 6.
    Boksa, P. Studies on the uptake and release of propranolol and the effects of propranolol on catecholamines in cultures of bovine adrenal chromaffin cells. Biochem. Pharmacol. 35: 805–815; 1986.PubMedCrossRefGoogle Scholar
  7. 7.
    Cavanaugh, M. W. Pulsation, migration and division in dissociated chicken embryo heart cells in vitro. J. Exp. Zool. 128: 573–589; 1955.CrossRefGoogle Scholar
  8. 8.
    Doupe, A. J.; Landis, S. C.; Patterson, P. D. Environmental influences: glucocorticoids, growth factors and chromaffin cell plasticity. J. Neurosci. 5: 2119–2142; 1985.PubMedGoogle Scholar
  9. 9.
    Furshpan, E. J.; Macleish, P. R.; O'Lague, P. H., et al. Chemical transmission between rat sympathetic neurons and cardiac myocytes developing in microcultures: evidence for cholinergic, adrenergic and dual-function neurons. Proc. Natl. Acad. Sci. USA 73: 4225–4229; 1976.PubMedCrossRefGoogle Scholar
  10. 10.
    Furshpan, E. J.; Landis, S. C.; Matsumoto, S. G., et al. Synaptic functions in rat sympathetic neurons in microcultures. I. Secretion of norepinephrine and acetylcholine. J. Neurosci. 6: 1061–1079; 1986.PubMedGoogle Scholar
  11. 11.
    Furshpan, E. J.; Potter, D. D.; Matsumoto, S. G. Synpatic functions in rat sympathetic neurons in microcultures. III. A purinergic effect on cardiac myocytes. J. Neurosci. 6: 1099–1107; 1986.PubMedGoogle Scholar
  12. 12.
    Georges, E.; Trifaró, J.-M.; Mushynski, W. E. Hypophosphorylated neurofilament subunits in the cytoskeletal and soluble fractions of cultured bovine adrenal chromaffin cells. Neuroscience 22: 753–763; 1987.PubMedCrossRefGoogle Scholar
  13. 13.
    Greene, L. A.; Rein, G. Release, storage and uptake of catecholamines by a clonal cell line of nerve growth factor (NGF) responsive pheochromocytoma cells. Brain Res. 129: 247–263; 1977.PubMedCrossRefGoogle Scholar
  14. 14.
    Harary, I.; Farley, B. In vitro studies of single isolated beating heart cells. Science 131: 1674–1675; 1960.PubMedCrossRefGoogle Scholar
  15. 15.
    Isenberg, G. Ca entry and contraction as studied in isolated bovine ventricular myocytes. Z. Naturforch. 37: 502–512; 1982.Google Scholar
  16. 16.
    Jacobson, S. L.; Piper, H. M. Cell cultures of adult cardiomyocytes as models of the myocardium. J. Mol. Cell. Cardiol. 18: 661–678; 1986.PubMedCrossRefGoogle Scholar
  17. 17.
    Kenigsberg, R. L.; Trifaro, J.-M. Presence of a high affinity uptake for catecholamines in cultured bovine adrenal chromaffin cells. Neuroscience 5: 1547–1556; 1980.PubMedCrossRefGoogle Scholar
  18. 18.
    King, K. L.; Boder, G. B., Williams, D. C., et al. Chronotropic effects of tyramine on rat heart cells cultured with sympathetic neurons. Eur. J. Pharmacol. 51: 331–335; 1978.PubMedCrossRefGoogle Scholar
  19. 19.
    Kirpekar, S. M.; Nobiletti, J.; Trifaró, J.-M. Effects of 6-hydroxydopamine on bovine adrenal chromaffin cells in culture. Br. J. Pharmacol. 9: 947–952; 1983.Google Scholar
  20. 20.
    Landis, S. C. Developmental changes in neurotransmitter properties of dissociated sympathetic neurons: a cytochemical study of the effects of medium. Dev. Biol. 77: 349–361; 1978.CrossRefGoogle Scholar
  21. 21.
    Lee, R. W. H.; Trifaró, J.-M. Characterization of an antibody against actin and its use in the study of the distribution of actin in cultured chromaffin cells. Neuroscience 6: 2087–2108; 1981.PubMedCrossRefGoogle Scholar
  22. 22.
    Libby, P. Long-term culture of contractile mammalian heart cells in a defined serum-free medium that limits non-muscle cell proliferation. J. Mol. Cell. Cardiol. 16: 803–811; 1984.PubMedCrossRefGoogle Scholar
  23. 23.
    Lieberman, M.; Roggeveen, A. E.; Purdy, J. E., et al. Synthetic strands of cardiac muscle: growth and physiological implication. Science 175: 909–911; 1975.CrossRefGoogle Scholar
  24. 24.
    Lieberman, M.; Sawanobori, T.; Kootsey, J. M., et al. A synthetic strand of cardiac muscle: its passive electrical properties. J. Gen. Physiol. 65: 527–550; 1975.PubMedCrossRefGoogle Scholar
  25. 25.
    Mark, G. E.; Strasser, F. E. Pacemaker activity and mitosis in cultures of newborn rat heart ventricle cells. Exp. Cell Res. 44: 217–233; 1966.PubMedCrossRefGoogle Scholar
  26. 26.
    Murray, M. R. Muscle. In: Willmer, E. N., ed. Cells and tissue in culture, methods, biology and physiology, vol. II, New York: Academic Press; 1975; 311–372.Google Scholar
  27. 27.
    Patrick, J.; Heinemann, S.; Schubert, D. Biology of cultured nerve and muscle. Ann. Rev. Neurosci. 1: 417–443; 1978.PubMedCrossRefGoogle Scholar
  28. 28.
    Patterson, P. H.; Chung, L. L. Y. The influence of non-neuronal cells on catecholamine and acetylcholine synthesis and accumulation in cultures of dissociated sympathetic neurons. Proc. Natl. Acad. Sci. USA 71: 3607–3610; 1974.PubMedCrossRefGoogle Scholar
  29. 29.
    Patterson, P. H.; Chun, L. Y. The induction of acetylcholine synthesis in primary cultures of dissociated rat sympathetic neurons. I. Effects of conditioned medium. Dev. Biol. 56: 263–280; 1977.PubMedCrossRefGoogle Scholar
  30. 30.
    Patterson, P. H.; Chun, L. Y. The induction of acetylcholine synthesis in primary cultures of dissociated rat symathetic neurons. II. Developmental aspects. Dev. Biol. 60: 473–481; 1977.PubMedCrossRefGoogle Scholar
  31. 31.
    Patterson, P. H. Environmental determination of autonomic neurotransmitter function. Ann. Rev. Neurosci. 1: 1–17; 1978.PubMedCrossRefGoogle Scholar
  32. 32.
    Potter, D. D.; Landis, S. C.; Matsumoto, S. G.; et al. Synaptic functions in rat sympathetic neurons in microcultures II. Adrenergic/cholinergic dual status plasticity. J. Neurosci. 6: 1080–1098; 1986.PubMedGoogle Scholar
  33. 33.
    Purves, R. D.; Hill, C. E.; Chamley, J. H., et al. Functional autonomic neuromuscular junctions in tissue culture. Pflugers Arch. 350: 1–7; 1974.PubMedCrossRefGoogle Scholar
  34. 34.
    Rinaldini, L. M. A quantitative method for growing animal cells in vitro. Nature 173: 1134–1135; 1954.PubMedCrossRefGoogle Scholar
  35. 35.
    Robinson, R. B. Models of cardiac development: transplants, organ culture, cell dispersion and cell culture. In: Legato, M. J., ed. The developing heart. Boston: Martinus Nijhoff Publishing; 1984: 69–94.Google Scholar
  36. 36.
    Schanne, O. F. Recording of contractile activity of cells in culture. J. Appl. Physiol. 29: 892–893; 1970.PubMedGoogle Scholar
  37. 37.
    Schanne, O. F. Factors involved in the loss of spontaneous contractile and electrical activity in clusters of cultured cardiac cells. Can. J. Physiol. Pharmacol. 50: 523–532; 1972.PubMedGoogle Scholar
  38. 38.
    Seraydarian, M. W.; Harary, I.; Sato, E. D. In vitro studies of beating-heart cells in culture XI. The ATP level and contractions of the heart cells. Biochem. Biophys. Acta 162: 414–423; 1968.PubMedCrossRefGoogle Scholar
  39. 39.
    Simpson, P.; Savion, S. Differentiation of rat myocytes in single cell cultures with and without proliferating nonmyocardial cells. Circ. Res. 50; 101–116; 1982.PubMedGoogle Scholar
  40. 40.
    Trifaró, J.-M.; Poisner, A. M.; Douglas, W. W. The fate of the chromaffin granule during catecholamine release from the adrenal medulla. I. Unchanged efflux of phospholipid and cholesterol. Biochem. Pharmacol. 16: 2095–2100; 1967.PubMedCrossRefGoogle Scholar
  41. 41.
    Trifaró, J.-M.; Ulpian, C. Isolation and characterization of myosin from the adrenal medulla. Neuroscience 1: 483–488; 1976.PubMedCrossRefGoogle Scholar
  42. 42.
    Trifaro, J.-M.. Common mechanisms of hormone secretion. Ann. Rev. Pharmacol. Toxicol. 17: 27–47; 1977.CrossRefGoogle Scholar
  43. 43.
    Trifaró, J.-M.; Ulpian, C.; Preiksaitis, H. Antimyosin stains chromaffin cells. Experientia 34: 1568–1571; 1978.PubMedCrossRefGoogle Scholar
  44. 44.
    Trifaró, J.-M.; Cubeddu, X. Exocytosis as a mechanism of noradrenergic transmitter release. In: Kalsner, S. ed. Trends in autonomic pharmacology, vol. I Baltimore, MD: Urban & Schwarzenberg; 1979: 195–249.Google Scholar
  45. 45.
    Trifaró, J.-M.; Lee, R. W. H. Morphological characteristics and stimulus-secretion coupling in bovine adrenal chromaffin cell cultures. Neuroscience 5: 1533–1546; 1980.PubMedCrossRefGoogle Scholar
  46. 46.
    Trifaró, J.-M. The cultured chromaffin cells: a model for the study of biology and pharmacology of paraneurons. Trends Pharmacol. Sci. 3: 389–392; 1982.CrossRefGoogle Scholar
  47. 47.
    Trifaró, J.-M.; Poisner, A. M. Common properties in the mechanisms of synthesis, processing and storage of secretory products. In: Poisner, A.; Trifaró, J.-M. eds. The secretory process, vol. I. North Holland: Elsevier Biochemical Press: 1982; 387–407.Google Scholar
  48. 48.
    Trifaró, J.-M. The adrenal paraneurone: its biology and pharmacology. Can. J. Physiol. Pharmacol. 62: 465–466; 1984.Google Scholar
  49. 49.
    Trifaró, J.-M.; Lee, R. W. H.; Puszkin, S. Immunofluorescent patterns of clathrin and dopamine-beta-hydroxylase in chromaffin cells in culture. Cell Tiss. Res. 235: 365–370; 1984CrossRefGoogle Scholar
  50. 50.
    Trifaró, J.-M.; Seward, E. P. Microinjection, techniques in the study of the secretory process. In: Poisner, A. M.; Trifaró, J.-M., eds. The secretory process, vol. III. New York: Elsevier Biomedical Press: 1987: 273–287.Google Scholar
  51. 51.
    Unsicker, K.; Chamley, J. H. Growth characteristics of postnatal rat adrenal medulla in culture. Cell Tiss. Res. 177: 247–268; 1977.CrossRefGoogle Scholar
  52. 52.
    Unsicker, K.; Krisch, B.; Otten, U. et al. Nerve growth factor-induced fiber outgrowth from isolated rat adrenal chromaffin cells: impairment by glucocorticoids. Proc. Natl. Acad. Sci. USA 75: 3498–3502; 1978.PubMedCrossRefGoogle Scholar
  53. 53.
    Vahouny, G. V.; Wei, R.; Starkweather, R. et al. Preparation of beating heart cells from adult rats. Science 167: 1616–1618; 1970.PubMedCrossRefGoogle Scholar
  54. 54.
    Vytásěk, R. A sensitive fluorometric assay for the determination of DNA Anal. Biochem. 120: 243–248; 1982.PubMedCrossRefGoogle Scholar
  55. 55.
    Wenzel, D. G.; Wheatley, J. W.; Don Byrd, G. Effects of nicotine on cultured rat heart cells. Toxicol. Appl. Pharmacol. 17: 774–785; 1970.PubMedCrossRefGoogle Scholar
  56. 56.
    Winkler, H.; Carmichael, S. W. The chromaffin granules. In: Poisner, A. M.; Trifaró, J.-M., eds. The secretory process, vol. I. The secretory granule. Amsterdam: Elsevier Biochemical: 1982; 3–79.Google Scholar
  57. 57.
    Wollenberger, A. Rhythmic and arrhythmic contractile activity of single myocardial cells cultured in vitro. Circ. res. 14–15 (Suppl II): 184–201; 1964.Google Scholar

Copyright information

© Tissue Culture Association 1990

Authors and Affiliations

  • J. M. Trifaró
    • 1
  • R. Tang
    • 1
  • M. L. Novas
    • 1
  1. 1.Secretory Process Research Program, Department of Pharmacology, Faculty of MedicineUniversity of OttawaOttawaCanada

Personalised recommendations