Skip to main content
SpringerLink
Log in

Alpha-1, alpha-2, and beta adrenergic signal transduction in cultured uterine myocytes

  • Regular Papers
  • Published:
In Vitro Cellular & Developmental Biology Aims and scope Submit manuscript

Summary

The following studies were undertaken to develop a cultured uterine myocyte model which would allow further clarification of the adrenergic signal transduction mechanisms utilized by these myocytes. After mechanical removal of the endometrium, rabbit uterine myoctes were isolated by an overnight enzymatic disaggregation using collagenase and DNase I. The isolated myocytes were maintained in culture in 75-cm2 flasks containing Waymouth's MB 751/1 medium-10% fetal bovine serum along with 10−8 M estradiol, penicillin, streptomycin, and Fungizone. The phase contrast and electron micrographic appearance of these cells was consistent with that previously reported for smooth muscle myocytes in culture. Immunocytochemical studies utilizing monoclonal anti-alpha-smooth muscle actin antibodies confirmed the presence of smooth muscle actin in these cultured myocytes. Western blot studies similarly confirmed the presence of alpha-smooth muscle actin in rabbit myometrial tissue and the cultured myocytes, both the primary and F1 generation. After prelabeling the myocytes with [3H]inositol, adrenergic stimulation experiments demonstrated alpha-1 receptor mediated stimulation of inositol phosphates. Beta receptor stimulation experiments confirmed cAMP production in these cultured myocytes, and the ability of clonidine, an alpha-2 agonist, to inhibit forskolin stimulated cAMP production confirmed the presence of functional alpha-2 adrenergic receptors in these myocytes. In conclusion, these cultured rabbit uterine myocytes have provided an in vitro model which can be utilized to further clarify the adrenergic receptor signal transduction mechanisms in genital tract smooth muscle.

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

Access this article

Log in via an institution

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

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Abdel-Latif, A. A.; Green, K.; Smith, J. P., et al. Norepinephrine-stimulate breakdown of triphosphoinositide of rabbit iris smooth muscle: effects of surgical sympathetic denervation and in vivo electric stimulation of the sympathetic nerve of the eye. J. Neurochem. 30:517–525; 1978.

    Article  PubMed  CAS  Google Scholar 

  2. Adelstein, R. S.; Eisenberg, E. Regulation and kinetics of the actin-myosin-ATP interactions. Ann. Rev. Biochem. 49:921–956; 1980.

    Article  PubMed  CAS  Google Scholar 

  3. Anwer, K.; Hovington, J. A.; Sanborn, B. M. Antagonism of contractants and relaxants at the level of intracellular calcium and phosphoinositide turnover in the rat uterus. Endocrinology 124:2995–3002; 1989.

    PubMed  CAS  Google Scholar 

  4. Berridge, M. J. Rapid accumulation of inositol trisphosphate reveals that agonist hydrolyse polyphosphoinositides instead of phosphatidylinositol. Biochem. J. 212:849–858; 1983.

    PubMed  CAS  Google Scholar 

  5. Boulet, A. P.; Fortier, M. A. Preparation and characterization of rabbit myometrial cells in primary culture: influence of estradiol and progesterone treatment. In Vitro Cell. Dev. Biol. 23:93–99; 1987.

    PubMed  CAS  Google Scholar 

  6. Boulet, A. P.; Fortier, M. A. Sex steroid regulation of β-adrenergic sensitive adenylate cyclase in rabbit myometrial cells in primary culture. Life Sci. 42:829–840; 1988.

    Article  PubMed  CAS  Google Scholar 

  7. Brown, E. M.; Hurwitz, S. H.; Aurbach, G. D. alpha-adrenergic inhibition of adenosine 3′,5′-monophosphate accumulation and parathyroid hormone release from dispersed bovine parathyroid cells. Endocrinology 103:893–899; 1978.

    PubMed  CAS  Google Scholar 

  8. Burgess, G. M.; McKinney, J. S.; Irvine, R. F., et al. Inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate formation in Ca2+-mobilizing-hormone-activated cells. Biochem. J. 232:237–243; 1985.

    PubMed  CAS  Google Scholar 

  9. Casey, M. L.; MacDonald, 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 

  10. Chamley, J. H.; Groschel-Stewart, U.; Campbell, G. R., et al. Distinction between smooth muscle, fibroblasts and endothelial cells in culture by use of fluoresceinated antibodies against smooth muscle actin. Cell Tissue Res 177:445–457; 1977.

    PubMed  CAS  Google Scholar 

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

    PubMed  CAS  Google Scholar 

  12. Fortier, M.; Chase, D.; Korenman, S. G., et al. β-adrenergic catecholamine-dependent properties of rat myometrium primary cultures. Am. J. Physiol. 245:C84-C90; 1983.

    PubMed  CAS  Google Scholar 

  13. Frielle, T.; Collins, S.; Daniel, K. W., et al. Cloning of the cDNA for the human B1-adrenergic receptor. Proc. Natl. Acad. Sci. USA 84:7920–7924; 1987.

    Article  PubMed  CAS  Google Scholar 

  14. Garcia-Sainz, J. A.; Hoffman, B. B.; Li, S. Y., et al. Role of alpha1 adrenoceptors in the turnover of phosphatidylinositol and of alpha2 adrenoceptors in the regulation of cyclic AMP accumulation in hamster adipocytes. Life Sci. 27:953–961; 1980.

    Article  CAS  Google Scholar 

  15. Hashimoto, T.; Hirata, M.; Ito, Y. A role for inositol 1,4,5-trisphosphate in the initiation of agonist-induced contractions of dog tracheal smooth muscle. Br. J. Pharmacol. 86:191–199; 1985.

    PubMed  CAS  Google Scholar 

  16. Hatjis, C. G. β-adrenergic-receptor and adenylate cyclase properties in pregnant and nonpregnant guinea pig myometrium. Am. J. Obstet. Gynecol. 151:943–950; 1985.

    PubMed  CAS  Google Scholar 

  17. Hayashida, D. N.; Leung, R.; Goldfien, A., et al. Human myometrial adrenergic receptors: identification of the betaadrenergic receptor by [3H]dihydroalprenolol binding. Am. J. Obstet. Gynecol. 142:389–393; 1982.

    PubMed  CAS  Google Scholar 

  18. Hoffman, B. B.; Lavin, T. N.; Lefkowitz, R. J., et al. Alpha adrenergic receptor subtypes in rabbit uterus: mediation of myometrial contraction and regulation by estrogens. J. Pharmacol. Exp. Ther. 219:290–295; 1981.

    PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  20. Hsu, C. J.; Sanborn, B. M. Relaxin affects the shape of rat myometrial cells in culture. Endocrinology 118:495–498; 1986.

    PubMed  CAS  Google Scholar 

  21. Hsu, C. J.; Sanborn, B. M. Relaxin treatment alters the kinetic properties of myosin light chain kinase activity in rat myometrial cells in culture. Endocrinology 118:499–505; 1986.

    PubMed  CAS  Google Scholar 

  22. Huszar, G.; Naftolin, F. The myometrium and uterine cervix in normal and preterm labor. N. Engl. J. Med. 311:571–581; 1984.

    Article  PubMed  CAS  Google Scholar 

  23. Joseph, S. K.; Thomas, A. P.; Williams, R. J., et al. Myo-inositol 1,4,5-trisphosphate. J. Biol. Chem. 259:3077–3081; 1984.

    PubMed  CAS  Google Scholar 

  24. Kao, H. W.; Finn, S. E.; Gown, A. M., et al. Cultured circular smooth muscle from the rabbit colon. In Vitro Cell. Dev. Biol. 24:787–794; 1988.

    PubMed  CAS  Google Scholar 

  25. Krall, J. F.; Morin, A. The role of cyclic AMP in cells with the properties of smooth muscle cultured from the rat myometrium. J. Cell. Physiol. 129:250–256; 1987.

    Article  Google Scholar 

  26. Krall, J. F. 17β-Estradiol-sensitivity of cultured myometrial cells. Experientia 43:608–610; 1987.

    Article  PubMed  CAS  Google Scholar 

  27. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685; 1980.

    Article  Google Scholar 

  28. Lefkowitz, R. J.; Caron, M. G.; Stiles, G. L. Mechanisms of membrane-receptor regulation. N. Engl. J. Med. 310:1570–1579; 1984.

    Article  PubMed  CAS  Google Scholar 

  29. Nabika, T.; Velletri, P. A.; Igawa, T., et al. Comparison of cyclic AMP accumulation and morphological changes induced by β-adrenergic stimulation of cultured vascular smooth muscle cells and fibroblasts. Blood Vessels 22:47–56; 1985.

    PubMed  CAS  Google Scholar 

  30. Norris, J. S.; Garmer, D. J.; Brown, F., et al. Characteristics of adenylate cyclase coupled beta2-adrenergic receptor in a smooth muscle tumor cell line. J. Receptor Res. 3:623–645; 1983.

    CAS  Google Scholar 

  31. Ohnishi, S. T.; Barr, J. K. A simplified method of quantitating protein using the biuret and phenol reagents. Anal. Biochem. 86:193; 1978.

    Article  PubMed  CAS  Google Scholar 

  32. Phillippe, M.; Saunders, T.; Hariharan, S. Absence of alpha-2 adrenergic effects of cAMP production in a genital tract smooth muscle cell line. Life Sci. 44:1555–1562; 1989.

    Article  PubMed  CAS  Google Scholar 

  33. Reynolds, E. E.; Dubyak, G. R. Activation of calcium mobilization and calcium influx by alpha1-adrenergic receptors in a smooth muscle cell line. Biochem. Biophys. Res. Comm. 130:627–632; 1985.

    Article  PubMed  CAS  Google Scholar 

  34. Richardson, M. R.; Taylor, D. A.; Casey, M. L., et al. Biochemical markers of contraction in human myometrial smooth muscle cells in culture. In Vitro Cell. Dev. Biol. 23:21–28; 1987.

    PubMed  CAS  Google Scholar 

  35. Riemer, R. K.; Goldfien, A.; Roberts, J. M. Estrogen increases adrenergic- but not cholinergic-mediated production of inositol phosphates in rabbit uterus. Mol. Pharmacol 32:663–668; 1987.

    PubMed  CAS  Google Scholar 

  36. Rifas, L.; Fant, J.; Makman, M. H., et al. The characterization of human uterine smooth muscle cells in culture. Cell Tissue Res. 196:385–395; 1979.

    Article  PubMed  CAS  Google Scholar 

  37. Schrey, M. F.; Cornford, P. A.; Read, A. M., et al. A role for phosphoinositide hydrolysis in human uterine smooth muscle during parturition. Am. J. Obstet. Gynecol. 159:964–970; 1988.

    PubMed  CAS  Google Scholar 

  38. Vallet-Strouve, C.; Mowszowicz, I. Myometrial cells in primary culture characterization and hormonal profile. Mol. Cell. Endocrinol. 12:97–110; 1978.

    Article  PubMed  CAS  Google Scholar 

  39. Wu, Y. Y.; Goldfien, A.; Roberts, J. M. Alpha adrenergic stimulation reduces cyclic adenosine 3′,5′-monophosphate generation in rabbit myometrium by two mechanisms. Biol. Reprod. 39:58–65; 1988.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine, University of Chicago, Pritzker School of Medicine, 60637, Chicago, Illinois

    Mark Phillippe, Trevania Saunders & Shrikar Bangalore

Authors
  1. Mark Phillippe
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Trevania Saunders
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shrikar Bangalore
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

This research was supported by grant HD-22063 from the National Institutes of Health, Bethesda, MD.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Phillippe, M., Saunders, T. & Bangalore, S. Alpha-1, alpha-2, and beta adrenergic signal transduction in cultured uterine myocytes. In Vitro Cell Dev Biol 26, 369–378 (1990). https://doi.org/10.1007/BF02623828

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02623828

Key words

Search

Navigation