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Fig. 2. Identification of NHE isoforms that correct acidosis in fully grown
oocytes. (Ai) RT-PCR of NHE isoforms in fully grown oocytes from
20-day-old mice. Amplicons formed by reverse transcription and PCR of positive
control tissues (+), three oocyte equivalents (O), or the final oocyte wash
drop (-) are shown for each isoform, as indicated. For further details see
Materials and methods. DNA ladder is in 100 bp increments. Distinct amplicons
were always generated from oocyte samples by NHE1, NHE3 and
NHE4 primers, but were never by NHE5 primers, and a very
faint band was produced by NHE2 primers using 40 cycles of PCR in
four of nine replicates. (Aii) RT-PCR of denuded oocytes from day-10
mice. Note that distinct amplicons were generated by primers for NHE1,
NHE3 and NHE4. Amplicons were not generated by primers for NHE2
or NHE5 (not shown). (B) Recovery from acidosis in mid-growth phase and
fully grown oocytes in bicarbonate-free medium. Note that, as was also the
case in the presence of bicarbonate (see
Fig. 1), only fully grown
oocytes recover form acidosis. (C) Typical examples of NH4Cl
pulse experiments on fully grown oocytes in bicarbonate-free media in the
presence of cariporide and S3226, as indicated. In each case, the drug was
added at t=20 minutes, and remained throughout the experiment unless
otherwise indicated. Note that 1 µM cariporide completely and reversibly
inhibited acidosis recovery. (D) Summary of all experiments performed
in this series plotted on a logarithmic scale. Drugs were diluted in DMSO such
that the final concentration of DMSO was 0.1% throughout. Note the break in
the x-axis to allow inclusion of a drug-free group (DMSO only). DMSO
alone did not influence the rate of recovery (P>0.5). Each data
point represents mean±s.d. of the initial rate of pH recovery from
three separate replicates, comprising between 41 and 26 oocytes.
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