Epithelial sodium conductance in rabbit preimplantation trophectodermal cells

Academic Article

Abstract

  • We examined the development of epithelial Na+ conductance in 6- and 7-day post coitus (p.c.) preimplantation rabbit embryos using the whole-cell patch-clamp technique on dissociated rabbit trophectodermal cells and by immunocytochemical localization using a polyclonal antibody directed against subunits of an apical epithelial Na+ channel on the intact blastocyst. In Day 6 and 7 p.c. trophectodermal cells, we observed an outwardly rectified whole-cell Na+ current. The current-voltage characteristics did not differ between the 6- and the 7-day p.c. cells. Replacement of Na+ with the impermeant cation N-methyl-d-glucamine in the pipette or bath reduced outward currents and inward currents, respectively, indicating that the current was Na+-dependent. Treatment of 7-day p.c. cells with 100 μM amiloride, benzamil, or ethylisopropyl amiloride (EIPA) blocked the whole-cell currents within 5 min. However, the current of the Day 6 p.c. embryo was not blocked by amiloride. The amiloride block at Day 7 p.c. was only partially reversible after 15 min of continuous perfusion of the bath with an amiloride-free solution. The apparent dissociation constant (Ki) for amiloride, benzamil, and EIPA was 12, 50, and 16 μM, respectively, when measured 5 min after drug addition. Immunolocalization studies of blastocysts with a polyclonal antibody raised against a high amiloride affinity Na+ channel isolated from bovine kidney revealed no specific binding to the trophectodermal cells at Day 6 p.c. At Day 7 p.c., however, an epitope, immunoreactive to the high amiloride affinity Na+ channel antibody, was predominantly localized to the microvilli on the apical membrane of the trophectodermal cells. These findings suggest that: (1) Day 6 and 7 p.c. trophectodermal cells have a Na+ conductance that displays similar current-voltage characteristics; (2) the apical expression of an epitope, possibly a protein related to an epithelial sodium channel, occurs between Days 6 and 7 p.c.; (3) an amiloride-sensitive component of the sodium current develops between Days 6 and 7 p.c.; and (4) the expression of immunoreactivity and development of amiloride sensitivity are temporally related. © 1991.
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    Author List

  • Robinson DH; Bubien JK; Smith PR; Benos DJ
  • Start Page

  • 313
  • End Page

  • 321
  • Volume

  • 147
  • Issue

  • 2