Skip to main content
Log in

Increase in cyclic AMP levels by relaxin in newborn rhesus monkey uterus cell culture

  • Rapid Communications in Cell Biology
  • Published:
In Vitro Cellular & Developmental Biology Aims and scope Submit manuscript

Editor's statement This paper details a smooth muscle cell line that is responsive to relaxin and provides a useful assay system for the hormone, as well as providing a model system for the study of the mechanisms of relaxin action.

Summary

A novel relaxin sensitive cell line of apparent smooth muscle origin has been established from a newborn rhesus monkey uterus (NRMU). NRMU cells respond to relaxin, in the presence of 1 μM forskolin, by producing intracellular adenosine 3′, 5′-cyclic monophosphate (cAMP). The increase in cAMP levels is dose, time and cell density dependent, reaching peak levels at 10 min when cells are seeded at 1×105 cells/well. Specificity was demonstrated by neutralization of the relaxin activity with anti-relaxin monoclonal and polyclonal antibodies, degradation of cAMP in the presence of phosphodiesterase, and confirmation of the absence of cGMP. Three synthetic analogs of human relaxin generated a dose-related cAMP response as did synthetic native human relaxin. Natural relaxin purified from human corpora lutea tissue also generated a response similar to synthetic human relaxin. Porcine and rat relaxins also increased levels of cAMP. Insulin, but not IGF I or IGF II, was capable of increasing cAMP levels in NRMU cells, however, 200 ng/mL were required to achieve cAMP levels comparable to 6.25 ng/ml relaxin. Combinations of relaxin with insulin, IGF I or IGF II did not increase cAMP levels above levels obtained with relaxin alone. The effect on NRMU cells of other hormones, growth factors and drugs potentially present in cell culture systems or serum samples was evaluated. In combination with relaxin, oxytocin significantly decreased the cAMP production below the levels induced by relaxin alone, whereas progesterone and prostaglandin E2 resulted in additive increases in cAMP. These data suggest that the NRMU cell line is an appropriate target tissue for studying relaxin-mediated biological responsesin vitro as well as functioning as the primary component of a relaxinin vitro bioassay.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

References

  1. Braddon, S. A. Relaxin-dependent adenosine 3′,5′-monophosphate concentration changes in the mouse pubic symphysis. Endocrinology 102:1292–1299; 1978.

    PubMed  CAS  Google Scholar 

  2. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248–254; 1976.

    Article  PubMed  CAS  Google Scholar 

  3. Casey, M. L.; Mac Donald, P. C.; Mitchell, M. D., et al. Maintenance and characterization of human myometrial smooth muscle cells in monolayer culture. In Vitro 20:396–403; 1984.

    Article  PubMed  CAS  Google Scholar 

  4. Chamley-Campbell, J.; Campbell, G.; Ross, R. The smooth muscle cell in culture. Physiol. Rev. 59:1–61; 1979.

    PubMed  CAS  Google Scholar 

  5. Charbord, P.; Gown, A. M.; Keating, A., et al. CGA-7 and HHF, two monoclonal antibodies that recognize muscle actin and react with adherent cells in human long-term bone marrow cultures. Blood 66:1138–1142; 1985.

    PubMed  CAS  Google Scholar 

  6. Cheah, S. H.; Sherwood, O. D. Target tissues for relaxin in the rat: tissue distribution of injected125I-labeled relaxin and tissue changes in adenosine 3′,5′-monophosphate levels after in vitro relaxin incubation. Endocrinology 106:1203–1209; 1980.

    PubMed  CAS  Google Scholar 

  7. Cole, W. C.; Garfield, R. E. Evidence for physiological regulation of myometrial gap junction permeability. Am. J. Physiol. 251 (Cell Physiol. 20):C411-C420; 1986.

    PubMed  CAS  Google Scholar 

  8. Drolet, D. W.; Henzel, W. J.; Johnson, P. D. Purification, amino-terminal sequencing and demonstration of biological activity of human relaxin from corpora lutea. 69th Annual Meeting, Endocrine Society, Indianapolis, IN, June 10 (Abstract 699) p. 196; 1987.

  9. Fager, G.; Hansson, G. K; Gown, A. M., et al. Human arterial smooth muscle cells in culture: inverse relationship between proliferation and expression of contractile proteins. In Vitro 25:511–520; 1989.

    CAS  Google Scholar 

  10. Gilman, A. G. A protein binding assay for adenosine 3′:5′-cyclic monophosphate. Proc. Natl. Acad. Sci. USA 67:305–312; 1970.

    Article  PubMed  CAS  Google Scholar 

  11. Gown, A. M.; Vogel, A. M.; Gordon, D., et al. A smooth muscle-specific monoclonal antibody that recognizes smooth muscle actin isoenzymes. J. Cell. Biol. 100:807–813; 1985.

    Article  PubMed  CAS  Google Scholar 

  12. Hsu, C. J.; McCormack, S. M.; Sanborn, B. M. The effect of relaxin on cyclic adenosine 3′,5′-monophosphate concentration in rat myometrial cells in culture. Endocrinology 116:2029–2035; 1985.

    PubMed  CAS  Google Scholar 

  13. Insel, P. A.; Bourne, H. R.; Coffino, P., et al. Cyclic AMP-dependent protein kinase: pivotal role in regulation of enzyme induction and growth. Science 190:896–898; 1975.

    Article  PubMed  CAS  Google Scholar 

  14. Johnston, P. D.; Burnier, J.; Chen, S., et al. Structure/Function studies on human relaxin. In: Deber, C. M.; Hruby, V. J.; Kopple, K. D., eds. Peptides: structure and function, proceedings of the Ninth American Peptide Symposium. 1985:683–686.

  15. Judson, D. G.; Pay, S.; Bhoola, K. D. Modulation of cyclic AMP in isolated rat uterine tissue slices by porcine relaxin. J. Endocrinol. 87:153–159; 1980.

    PubMed  CAS  Google Scholar 

  16. Kearney, J. F.; Radbruch, A.; Liesegang, B., et al. A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines. J. Immunol. 123:1548–1550; 1979.

    PubMed  CAS  Google Scholar 

  17. Kemp, B. E.; Niall, H. D. Relaxin. In: Aurbach, G. D.; McCormick, D. B., eds. Vitamins and hormones. New York: Academic Press; 41:79–114; 1984.

    Google Scholar 

  18. Korenman, S. G.; Bhalla, R. C.; Sanborn, B. M., et al. Protein kinase translocation as an early event in the hormonal control of uterine contraction. Science 1983:430–432; 1974.

    Article  Google Scholar 

  19. Korenman, S. G.; Krall, J. F. The role of cyclic AMP in the regulation of smooth muscle contraction in the uterus. Biol. Reprod. 16:1–17; 1977.

    Article  PubMed  CAS  Google Scholar 

  20. Kuo, J. F.; Greengard, P. Cyclic nucleotide-dependent protein kinases. IV. Widespread occurrence of adenosine 3′,5′-monophosphate-dependent protein kinase in various tissues and phyla of the animal kingdom. Proc. Natl. Acad. Sci. USA 64:1349–1355; 1969.

    Article  PubMed  CAS  Google Scholar 

  21. Leroy, M. J.; Ferre, F.; Filliatreau, G., et al. Cyclic AMP metabolism in the inner and outer layers of the human myometrium near term. Acta. Physiologica. Hungarica. 67:83–94; 1986.

    PubMed  CAS  Google Scholar 

  22. Littlefield, J. W. Selection of hybrids from matings of fibroblastsin vitro and their presumed recombinants. Science 145:709–710; 1964.

    Article  PubMed  CAS  Google Scholar 

  23. Luben, R. A.; Chen, M. C-Y.; Rosen, D. M., et al. Effects of osteoclast activating factor from human lymphocytes on cyclic AMP concentration in isolated mouse bone and bone cells. Calcif. Tissue Int. 28:23–32; 1979.

    Article  PubMed  CAS  Google Scholar 

  24. Lucas, C.; Bald, L. N.; Martin, M. C., et al. An enzyme-linked immunosorbent assay to study human relaxin in human pregnancy and in pregnant rhesus monkeys. J. Endocrinol. 120:449–457; 1989.

    Article  PubMed  CAS  Google Scholar 

  25. Moyle, W. R.; Kong, Y. C.; Ramachandran, J. Steroidogenesis and cyclic adenosine 3′,5′-monophosphate accumulation in rat adrenal cells. J. Biol. Chem. 248:2409–2417; 1973.

    PubMed  CAS  Google Scholar 

  26. Oi, V.; Herzenberg, L. Immunoglobulin-producing hybrid cell lines. In: Mishel, B.; Schiigi, S., eds. Selected Methods In Cellular Immunology. San Francisco, CA: W. J. Freeman Co.; 1980:351–372.

    Google Scholar 

  27. Partridge, N. C.; Kemp, B. E.; Livesey, S. A., et al. Activity ratio measurements reflect intracellular activation of adenosine 3′,5′-monophosphate-dependent protein kinase in osteoblasts. Endocrinology 111:178–183; 1982.

    PubMed  CAS  Google Scholar 

  28. Potter, M.; Humphrey, J. G.; Walters, J. L. Growth of primary pharmacytomas in the mineral oil conditioned peritoneal environment. J. Natl. Cancer Inst. 49:305–308; 1972.

    PubMed  CAS  Google Scholar 

  29. Sanborn, B. M.; Bhalla, R. C.; Korenman, S. G. The endometrial adenosine cyclic 3′,5′-monophosphate-dependent protein kinase. J. Biol. Chem. 248:3593–3600; 1973.

    PubMed  CAS  Google Scholar 

  30. Sanborn, B. M.; Korenman, S. G. Further studies of the interaction of cyclic adenosine 3′,5′-monophosphate with endometrial protein kinase. J. Biol. Chem. 248:4713–4715; 1973.

    PubMed  CAS  Google Scholar 

  31. Sanborn, B. M.; Kuo, H. S.; Weisbrodt, N. W., et al. The interaction of relaxin with the rat uterus I. Effect on cyclic nucleotide levels and spontaneous contractile activity. Endocrinology 106:1201–1215; 1980.

    Article  Google Scholar 

  32. Sanborn, B. M.; Heindel, J. J.; Robinson, G. A. The role of cyclic nucleotides in the reproductive processes. Ann. Rev. Physiol. 42:37–57; 1980.

    Article  CAS  Google Scholar 

  33. Seamon, K. B.; Padgett, W.; Daly, J. W. Forskolin: unique diterpene activator of adenylate cyclase in membranes and in intact cells. Proc. Natl. Acad. Sci. USA 78:3363–3367; 1981.

    Article  PubMed  CAS  Google Scholar 

  34. St. Louis, J. Relaxin inhibition of KCl-induced uterine contractionsin vitro: an alternative bioassay. Can. J. Physiol. Pharmacol. 59:507–512; 1981.

    PubMed  CAS  Google Scholar 

  35. Steinetz, B. G.; Beach, V. L.; Kroc, R. L., et al. Bioassay of relaxin using a reference standard: a simple and reliable method utilizing direct measurement of interpubic ligament formation in mice. Endocrinology 67:102–115; 1960.

    PubMed  CAS  Google Scholar 

  36. Sternberger, L. A. Immunocytochemistry, 2nd ed, John Wiley and Sons, New York; 1979.

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kramer, S.M., Gibson, U.E.M., Fendly, B.M. et al. Increase in cyclic AMP levels by relaxin in newborn rhesus monkey uterus cell culture. In Vitro Cell Dev Biol 26, 647–656 (1990). https://doi.org/10.1007/BF02624216

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02624216

Key words

Navigation