The Journal of Membrane Biology

, Volume 194, Issue 3, pp 165–176 | Cite as

Regulation of Ion Fluxes, Cell Volume and Gap Junctional Coupling by cGMP in GFSHR-17 Granulosa Cells

  • A. Ngezahayo
  • B. Altmann
  • H. -A. KolbEmail author


Gap junctional communication between granulosa cells seems to play a crucial role for follicular growth and atresia. Application of the double whole-cell patch-clamp- and ratiometric fura-2-techniques allowed a simultaneous measurement of gap junctional conductance (G j) and cytoplasmic concentration of free Ca2+ ([Ca2+]i) in a rat granulosa cell line GFSHR-17. The voltage-dependent gating of G j varied for different cell pairs. One population exhibited a bell-shape dependence of G j on transjunctional voltage, which was strikingly similar to that of Cx43/Cx43 homotypic gap junction channels expressed in pairs of oocytes of Xenopus laevis. Within 15–20 min, gap junctional uncoupling occurred spontaneously, which was preceded by a sustained increase of [Ca2+]i and accompanied by shrinkage of cellular volume. These responses to the whole-cell configuration were avoided by absence of extracellular Ca2+, blockage of K+ efflux, or addition of 8-bromoguanosine 3′,5′-cyclic monophosphate (8-Br-cGMP) to the pipette solution. Even in the absence of extracellular Ca2+ or blockage of K+ efflux, formation of whole-cell configuration generated a Ca2+ spike that could be suppressed by the presence of 8-Br-cGMP. We propose that intracellular cGMP regulates Ca2+ release from intracellular Ca2+ stores, which activates sustained Ca2+ influx, K+ efflux and cellular shrinkage. We discuss whether gap junctional conductance is directly affected by cGMP or by cellular shrinkage and whether gap junctional coupling and/or cell shrinkage is involved in the regulation of apoptotic/necrotic processes in granulosa cells.


Granulosa cells Gap junctions Ca2+ Cell volume shrinkage cGMP Apoptosis 



The authors thank Dr. B. Sommersberg, Dr. A. Mayerhofer, Anatomisches Institut, Technische Universität München, Germany, and Dr. A. Amsterdam, Weizman Institute Rehevot, Israel, for providing the GFSHR-17 cell line. The work was partly supported by a grant of the Fritz Thyssen-Stiftung.


  1. 1.
    Ackert, C.L., Gittens, J.E.I., O'Brien, M.J., Eppig, J.J., Kidder, G.M. 2001Intercellular communication via connexin43 gap junctions is required for ovarian folliculogenesis in the mouse.Dev. Biol.233248270CrossRefGoogle Scholar
  2. 2.
    Banach, K., Weingart, R. 1996Connexin43 gap junctions exhibit asymmetrical gating properties.Pfluegers Arch.431775785CrossRefGoogle Scholar
  3. 3.
    Bevans, C.G., Kordel, M., Rhee, S.K., Harris, A.L. 1998Isoform composition of connexin channels determines selectivity among second messengers and uncharged molecules.J. Biol. Chem.27328082816CrossRefPubMedGoogle Scholar
  4. 4.
    Bortner, C.D., Hughes Jr., A.F.M., Cidlowski, J.A. 1997primary role for K+ and Na+ efflux in the activation of apoptosis.J. Biol. Chem.2723243632442CrossRefPubMedGoogle Scholar
  5. 5.
    Bortner, C.D., Cidlowski, J.A. 1999Caspase independent/dependent regulation of K+, cell shrinkage, and mitochondrial membrane potential during lymphocyte apoptosis.J. Biol. Chem.2742195321962CrossRefPubMedGoogle Scholar
  6. 6.
    Bruzzone, R., White, T.W., Paul, D.L. 1996Connections with connexins: the molecular basis of direct intercellular signaling.Eur. J. Biochem.238127PubMedGoogle Scholar
  7. 7.
    Chen-Izu, Y., Moreno, A.P., Spangler, R.A. 2001Opposing gates model for voltage gating of gap junctional channels.Am. J. Physiol.281C1604C1613Google Scholar
  8. 8.
    Dallaporta, B., Hirsch, T., Susin, S.A., Zamzami, N., Larochette, N., Brenner, C., Marzo, I., Kroemer, G. 1998Potassium leakage during the apoptotic degradation phase.J. Immunol.16056055615PubMedGoogle Scholar
  9. 9.
    Elenes, S., Martinez, A.D., Delmar, M., Beyer, E.C., Moreno, A.P. 2001Heterotypic docking of Cx43 and Cx45 connexons blocks fast voltage gating of Cx43.Biophys. J.8114061418PubMedGoogle Scholar
  10. 10.
    Estévez, A.G., Spear, N., Thompson, J.A., Cornwell, T.L., Radi, R., Barbeito, L., Beckman, J.S. 1998Nitric oxide-dependent production of cGMP supports the survival of rat embryonic motor neurons cultured with brain-derived neurotrophic factor.J. Neurosci.1837083714PubMedGoogle Scholar
  11. 11.
    Godwin, A.J., Green, L.M., Walsh, M.P., McDonald, J.R., Walsh, D.A., Fletcher, W.H. 1993In situ regulation of cell-cell communication by the cAMP-dependent protein kinase and protein kinase C.Mol. Cell Biochem.127-128293307PubMedGoogle Scholar
  12. 12.
    Gómez-Angelats, M., Bortner, C.D., Cidlowski, J.A. 2000Cell volume regulation in immune cell apoptosis.Cell Tissue Res.3013342CrossRefPubMedGoogle Scholar
  13. 13.
    Goodenough, D.A., Simon, A.M., Paul, D.L. 1999Gap junctional intercellular communication in the mouse ovarian follicle.Cardew, . eds. Gap junction-mediated intercellular signalling in health and disease. Novartis foundation,John Wiley & SonsChichester, New York, Weinheim, Brisbane, Toronto, Singapore226240Google Scholar
  14. 14.
    Grynkiewicz, G., Poenie, M., Tsien, R.Y. 1985A new generation of Ca2+ indicators with greatly improved fluorescence properties.J. Biol. Chem.260344050PubMedGoogle Scholar
  15. 15.
    Gutkowska, J., Jankowski, M., Sairam, M.R., Fujio, N., Reis, A.M., Mukaddam-Daher, S., Tremblay, J. 1999Hormonal regulation of natriuretic peptide system during induced follicular development in the rat.Biol. Repr.61162170Google Scholar
  16. 16.
    Harris, A.L. 2001Emerging issues of connexin channels: biophysics fills the gap.Q. Rev. Biophys.34325472PubMedGoogle Scholar
  17. 17.
    Hirsch, J.R., Weber, G., Kleta, L., Schlatter, E. 1999A novel cGMP-regulated K+ channel in immortalized human kidney epithelial cell (IHKE-1).J. Physiol.519645655PubMedGoogle Scholar
  18. 18.
    Kanemitsu, M.Y., Jiang, W., Eckhart, W. 1998Cdc2-mediated phosphorylation of the gap junction protein, connexin43, during mitosis.Cell Growth Differ.91321PubMedGoogle Scholar
  19. 19.
    Keren, T.I., Dantes, A., Sprengel, R., Amsterdam, A. 1993Establishment of steroidegenic granulosa cell lines expressing follicle stimulating hormone receptors.Mol. Cell Endocrinol.95R1R10CrossRefPubMedGoogle Scholar
  20. 20.
    Krick, S., Platoshyn, O., Sweeney, M., Kim, H., Yuan, J.X.-J. 2001Activation of K+ channels induces apoptosis in vascular smooth muscle cells.Am. J. Physiol.280C970C979Google Scholar
  21. 21.
    Maeno, E., Ishizaki, Y., Kanazeki, T., Hazama, A., Okada, Y. 2000Normotonic cell shrinkage because of disordered volume regulation is an early prerequisite to apoptosis.Proc. Natl. Acad. Sci. USA9794879492CrossRefPubMedGoogle Scholar
  22. 22.
    McGee, E., Spears, N., Minami, S., Hsu, S.-Y., Chung, Y.-Y., Billig, H., Hsueh, A.J.W. 1997Preantral ovarian follicles in serum-free culture: suppression of apoptosis after activation of cyclic guanosine 3′,5′-moonophosphate pathway and stimulation of growth and differentiation by follicle-stimulating hormone.Endocrinology13824172424PubMedGoogle Scholar
  23. 23.
    Moreno, H., Vega-Saena de Miera, E., Nadal, M.S., Amarillo, Y., Rody, B. 2001Modulation of Kv3 potassium channels expressed in CHO cells by a nitric oxide-activated phosphatase.J. Physiol.530345358PubMedGoogle Scholar
  24. 24.
    Neyton, J., Trautmann, A. 1985Single-channel currents of an intercellular junction.Nature317331335PubMedGoogle Scholar
  25. 25.
    Ngezahayo, A., Kolb, H.-A. 1990Gap junctional permeability is affected by volume changes and modulates volume regulation.FEBS Lett.27668CrossRefPubMedGoogle Scholar
  26. 26.
    Ngezahayo, A., Kolb, H.-A. 1994Regulation of gap junctional coupling in isolated pancreatic acinar cell pairs by cholecystokinin-octapeptide, vasoactive intestinal peptide (VIP) and a VIP-antagonist.J. Membrane Biol.139127136Google Scholar
  27. 27.
    Ngezahayo, A., Zeilinger, C., Todt, L., Marten, I., Kolb, H.-A. 1998Inactivattion of expressed and conducting rCx46 hemichannels by phosphorylation.Pfluegers Arch.436627629CrossRefGoogle Scholar
  28. 28.
    Noubani, A., Farookhi, R., Gutkowska, J. 2000B-type natriuretic peptide receptor expression and activity are hormonally regulated in rat ovarian cells.Endocrinology141551559PubMedGoogle Scholar
  29. 29.
    Okuma, A., Kuraoka, A., Iida, H., Inai, T., Wasano, K., Shibata, Y. 1996Colocalization of connexin 43 and connexin 45 but absence of connexin 40 in granulosa cell gap junctions of rat ovary.J. Reprod. Fert.107255264Google Scholar
  30. 30.
    Perez, G.I., Maravei, D.V., Trbovich, A.M., Cidlowski, J.A., Tilly, J.L., Hughes Jr., F.M. 2000Identification of potassium-dependent and -independent components of the apoptotic machinery in mouse ovarian germ cells and granulosa cells.Biol. Reprod.6313581369PubMedGoogle Scholar
  31. 31.
    Schreiber, J.R., Beckmann, M.W., Polacek, D., Davis, P.F. 1993Changes in gap junction connexin-43 messenger ribonucleic acid levels associated with rat follicular development as demonstrated by in situ hybridization.Am. J. Obstet. Gynecol.16810942204PubMedGoogle Scholar
  32. 32.
    Sommersberg, B., Bulling, A., Salzer, U., Frohlich, U., Garfield, R.E., Amsterdam, A., Mayerhofer, A. 2000Gap junction communication and connexin 43 gene expression in a rat granulosa cell line: regulation by follicle-stimulating hormone.Biol. Reprod.6316611668PubMedGoogle Scholar
  33. 33.
    Somogyi, R., Batzer, A., Kolb, H.-A. 1989Inhibition of electrical coupling in pairs of murine pancreatic acinar cells by OAG and isolated protein kinase C.J. Membrane Biol.108273282Google Scholar
  34. 34.
    Sosinsky, G. 1995Mixing of connexins in gap junction membrane channels.Proc. Natl. Acad. Sci. USA9292109214PubMedGoogle Scholar
  35. 35.
    Steiner, E., Ebihara, L. 1996Functional characterization of canine connexin45.J. Membrane Biol.150153161CrossRefGoogle Scholar
  36. 36.
    Stergiopoulos, K., Alvarado, J.L., Mastroianni, M., Ek-Vitorin, J.F., Taffet, S.M., Delmar, M. 1999Hetero-domain interactions as a mechanism for the regulation of connexin channels.Circ. Res.8411441155PubMedGoogle Scholar
  37. 37.
    Takuma, K., Phuagphong, P., Lee, E., Mori, K., Baba, A., Matsuda, T. 2001Anti-apoptotic effect of cGMP in cultured astrocytes.J. Biol. Chem.2764809348099PubMedGoogle Scholar
  38. 38.
    Tilly, J.L. 1996Apoptosis in ovarian function.Rev. Reprod.1162172PubMedGoogle Scholar
  39. 39.
    Todt, I., Ngezahayo, A., Ernst, A., Kolb, H.-A. 2001Hydrogen peroxide inhibits gap junctional coupling and modulates intracellular free calcium in cochlear Hensen cells.J. Membrane Biol.181107114Google Scholar
  40. 40.
    Veenstra, R. 2001Voltage clamp limitations of dual whole-cell gap junction current and voltage recordings. I. Conductance measurements.Biophys. J.8022312247PubMedGoogle Scholar
  41. 41.
    Veenstra, R.D., Wang, H.Z., Beyer, E.C., Brink, P.R. 1994Selective dye and ionic permeability of gap junction channels formed by connexin45.Circ. Res.75483489PubMedGoogle Scholar
  42. 42.
    White, T.W., Bruzzone, R. 1996Multiple connexin proteins in single intercellular channels: connexin compatibility and functional consequences.J. Bioenerg. Biomembr.28339350PubMedGoogle Scholar
  43. 43.
    White, T.W., Paul, D.L. 1999Genetic diseases and gene knockouts reveal diverse connexin functions.Annu. Rev. Physiol.61283310CrossRefPubMedGoogle Scholar
  44. 44.
    Wiesen, J.F., Midgley, R.A. 1994Expression of connexin43 gap junction messenger ribonucleic acid and protein during follicular atresia.Biol. Reprod.50336348PubMedGoogle Scholar
  45. 45.
    Willecke, K., Eiberger, J., Degen, J., Eckardt, D., Romualdi, A., Guldenagel, M., Deutsch, U., Sohl, G. 2002Structural and functional diversity of connexin genes in the mouse and human genome.Biol. Chem.383725737PubMedGoogle Scholar
  46. 46.
    Wright, C.S., Becker, D.L., Lin, J.S., Warner, A.E., Hardy, K. 2001Stage-specific and differential expression of gap junctions in the mouse ovary: connexin-specific roles in follicular regulation.Reproduction1217788PubMedGoogle Scholar
  47. 47.
    Zhao, H.B., Santos-Sacchi, J. 1998Effect of membrane tension on gap junctional conductance of supporting cells in Corti's Organ.J. Gen. Physiol.112447455CrossRefPubMedGoogle Scholar
  48. 48.
    Zhao, H.B., Santos-Sacchi, J. 2000Voltage gating of gap junctions in cochlear supporting cells: evidence for nonhomotypic channels.J. Membrane Biol.1751724CrossRefGoogle Scholar

Copyright information

© Springer-Verlag New York Inc. 2003

Authors and Affiliations

  1. 1.Institut für BiophysikUniversität Hannover, Herrenhäuserstr. 2, D-30419 HannoverGermany

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