Fig. 4. Identification of NHE isoforms that correct acidosis in granulosa shells. (A) RT-PCR of NHE isoforms in granulosa shells isolated from 10-day-old mice. For each isoform, products formed by reverse transcription and PCR of positive control tissues (+), one follicle-equivalent of granulosa cells (G), or oocyte bathing media (-) are shown. For further details see Materials and methods. (B) Acidosis recovery in granulosa cells in bicarbonate-free medium. Note that amiloride retards the Na⁺-dependent pH_i increase that occurs under control conditions. (C) Examples of NH₄Cl pulse experiments of granulosa shells in bicarbonate-free medium in the presence of cariporide and S3226 (as indicated in each panel). In each case, the drug was added at t=10 minutes, and remained throughout the experiment. (D) Summary of all experiments performed in this series. DMSO (vehicle) was 0.1% throughout. DMSO alone had no effect upon acidosis recovery. Since some recovery occurs during the Na⁺-free period in granulosa cells in these experiments, the rate of pH_i increase during the Na⁺-free period was subtracted to obtain the Na⁺-dependent component of recovery. Each data point represents mean±s.d. of the Na⁺-dependent pH_i recovery from three to five separate replicates, comprising between 18 and 32 granulosa shells. The results of the amiloride and the 10 µM cariporide + 1 µM S3226 co-treatment experiments have been added as a bar graph for ease of comparison.