Study of fibroblast spreading: pH dependence, involvement of the Na+/H(+)-antiporter and PKC

The pH dependence of spreading of normal mouse embryo fibroblasts was investigated. It was shown that, in contrast to DNA synthesis, cell spreading did not depend on intracellular pH in the same pH range. Serum growth factors had no influence on intracellular pH in the suspended cells. Spreading of the fibroblasts on poly-L-lysine-coated coverslips in a serum-free medium did not alter intracellular pH. Cytoplasm alkalynization accompanying the fibroblasts spreading in a medium with serum is suggested to be due to the effect of serum growth factors on the spread cells. The inhibitor of the Na+ H(+)-antiporter, MIBA, and the inhibitor of PKC, staurosporin, as well as the PMA-induced cell depletion of PKC prevented pH increase, but had no effect on the spreading itself. It is concluded that the pH increase observed during fibroblasts spreading in a serum-containing medium is not required for the spreading itself being due to the activation of both the Na+/H(+)-antiporter and PKC.