Estimating intracellular pH in developing rodent embryos using a computer imaging technique: changes in embryonic pH and proliferation rates following maternal treatment with acetazolamide.

Abstract

Using the transplacental distribution of the weak acid 5,5-dimethyloxazolidine-2,4-dione (DMO), a computer assisted imaging technique has been developed to permit the estimation of intracellular pH (pHi) in very specific areas of the developing rodent embryo. The study reported here demonstrates the heterogeneity of radiolabeled DMO distribution in the developing mouse forelimb. The pattern of pHi distribution shifts from one of high pHi values in the proximal core of the mesoderm on day 10 of gestation to one of higher pHi values in the mesoderm just underlying the ectoderm on day 11. Studies [Scott et al. (1990) Toxicol. Appl. Pharmacol. 103:238-254] in which DMO concentration was monitored following treatment with acetazolamide or acetazolamide plus amiloride were done in whole embryo homogenates or pooled limb samples which allow for the calculation of an average pHi but may not reflect the pHi in very specific locations of the limb. Two hours after acetazolamide administration, the pHi pattern was not significantly changed from control. Intracellular pH was raised above control levels but was not significant statistically except in the peripheral mesoderm in the ventral third of the forelimb. Fifteen hours after acetazolamide treatment, there was a significant decrease in pHi values with no change in pattern. However, treatment with acetazolamide plus amiloride for 15 hr produced a marked reduction of pHi values throughout the forelimb bud. Changes in bromodeoxyuridine labeling index (an indication of proliferative activity) following treatment with acetazolamide or acetazolamide plus amiloride are reported. The combination treatment reduced the labeling index by approximately 15% below that of control embryos in the limb region where absence of digit(s) will occur. However, we found no overall correlation of proliferative rate and pHi of limb bud mesoderm in treated embryos. Consequently, we were unable to causally associate reduced pHi with decreased proliferative rate.