Effects of hypotonic shock on intracellular pH in bovine articular chondrocytes

Chondrocytes inhabit an unusual environment, in which they are repeatedly subjected to osmotic challenges as fluid is expressed from the extracellular matrix during static joint loading. In the present study, the effects of hypotonic shock on intracellular pH, pH(i), have been studied in isolated bovine articular chondrocytes using the pH-sensitive fluroprobe BCECF. Cells subjected to a 50% dilution rapidly alkalinised, by approximately 0.2 pH units, a sustained plateau being achieved within 300 s. The effect was not altered by inhibitors of pH regulators, such as amiloride, bafilomycin and SITS, but was absent when cells were subjected to hypotonic shocks in solutions in which Na(+) ions were replaced by NMDG(+). The response was found to be sensitive to Gd(3+) ions, blockers of stretch-activated cation channels. Alkalinisation was also inhibited by treatment with Zn(2+) ions, at a concentration reported to block voltage-activated H(+) channels (VAHC). Depolarisation using high K(+) solutions supplemented with valinomycin also induced intracellular alkalinisation. Measurements using a membrane potential (E(m)) fluorescent dye showed that E(m) was approximately -44 mV, but was depolarised by over 50 mV following HTS. The depolarisation was also inhibited by Na(+) substitution with NMDG(+) or treatment with Gd(3+). We conclude that in response to HTS the opening of a stretch-activated cation channel leads to Na(+) influx, which results in a membrane depolarisation. Subsequent activation of VAHC permits H(+) ion efflux along the prevailing electrochemcial gradient, leading to the alkalinisation, which we record.