Modulation of Na+ x H+ exchange by hydrostatic pressure in isolated bovinearticular chondrocytes

The effects of increased hydrostatic pressure on Na+ x H+ exchange activity in bovine articular chondrocytes have been characterized. Chondrocytes wer e belated from the cartilage matrix and the cells were loaded with the pH-s ensitive fluorophore BCECF. Cells were acidified by ammonium rebound and th e rate of recovery of pH(i) back to control levels was determined using cuv ette fluorimetry. The application of hydrostatic pressure (1-300 atm) to ce lls within the fluorimeter was found to stimulate the rate of recovery from acidification, recorded as proton fluxes, in MOPS buffered media. This inc rease was dependent on the presence of extracellular Na+ ions and was inhib ited by the Na+ x H+ exchange inhibitor EIPA. The pressure-stimulated incre ase in H+ flux is therefore mediated completely by Na+ x H+ exchange. In ad dition, the stimulation could be abolished by the kinase inhibitor staurosp orine, was not additive with the stimulation of Na+ x H+ exchange elicited by the addition of serum and was unaffected by low concentrations of the my osin light chain kinase inhibitor ML-7. We therefore conclude that hydrosta tic pressure activates Na+ x H+ exchange in this cell type by a pathway whi ch involves direct phosphorylation of the transporter protein itself. This is the fi rst demonstration of the activation of Na+ x H+ exchange by hydro static pressure and the relevance of this finding to the biology of cartila ge tissue is discussed.