Postaxial forelimb
ectrodactyly induced by
acetazolamide given on Day 9.5 of murine gestation is thought to be mediated by reduced intracellular pH (pHi) within the limb bud. Coadministration of
amiloride increases the incidence and severity of
acetazolamide-induced forelimb malformations and further reduces limb bud pHi. These findings were hypothesized to be attributable to the action of
amiloride as an inhibitor of Na+/H+ exchangers (NHEs), plasma membrane-localized
proteins involved in the maintenance of cellular pH homeostasis. Here, we explored this hypothesis further by coadministering with
acetazolamide,
amiloride, or analogs known to preferentially inhibit NHEs 5-(N-methyl-N-isobutyl)-amiloride, 5-(N, N-hexamethylene)-amiloride, 5-(N, N-dimethyl)-amiloride, and 5-(N-ethyl-N-isopropyl)-amiloride or
amiloride-sensitive Na+ channels (
benzamil). The coadministration of either
amiloride,
benzamil, 5-(N, N-dimethyl)-amiloride, 5-(N-ethyl-N-isopropyl)-amiloride, or 5-(N-methyl-N-isobutyl)-amiloride all dose responsively increased the frequency and severity of forelimb malformations compared to
acetazolamide alone. None of the analogs given alone induced forelimb
ectrodactyly. The data are consistent with the original hypothesis that the exacerbation of
acetazolamide teratogenesis is due to NHE inhibition. Surprisingly,
benzamil was the most potent potentiator of
acetazolamide teratogenesis. This result strongly suggests that
amiloride-sensitive Na+ channels are also present within the murine embryo and are likely to play a role in pHi homeostasis.