Measurement of intracellular pH in hamster diaphragm by absorption spectrophotometry

The regulation of intracellular pH (pHi) is important in controlling muscle contraction. In these experiments, a spectrophotometric method of determining pHi was developed, and the method was then used to study muscle pHi regulation during CO2-induced changes in extracellular pH (pHb). Studies were performed in vitro on 27 diaphragm muscle strips obtained from adult hamsters. pHi was measured from the ratio of the absorbances of the acid (lambda = 530 nm) and alkaline (lambda = 460 nm) forms of a vital dye, neutral red, using the unstained diaphragm spectrum as a reference blank. A standard neutral red calibration curve constructed from eight diaphragm muscle homogenates indicated that the absorbance ratio was highly linear, with pH over the range 6.00-8.00. In intact muscle strips gassed with 95% O2-5% CO2, pHb was 7.45 +/- 0.03 (SE) and pHi was 7.00 +/- 0.01 (SE). When the muscle was aerated with CO2 concentrations from 3 to 30%, pHb and pHi changed rapidly and reached a steady state in 10-15 min. However, when pHb ranged from 6.80-7.80, pHi changed little from the value observed when pHb was 7.40. When pHb was less than 6.80 or greater than 7.80, changes in pHi and pHb were quantitatively similar. The results suggest that, in the isolated diaphragm, overall pHi is stable and effectively buffered over a wide range of CO2-induced changes in buffer solution pH.