Cation dependence of guinea pig epidermal cell spreading

To understand the earliest phases of epidermal cell spreading we have sought a defined in vitro system. We studied the divalent cation dependence of guinea pig epidermal cell spreading in media containing varying concentrations of cations. No spreading occurred in calcium-magnesium-free Dulbecco's modified Eagle's medium (CMF-DME) in the presence of cation-free fetal bovine serum; however, significant spreading occurred if the medium was supplemented with Mg++ plus Ca++ or Mg++ alone. Supplementing with Ca++ alone led to much less spreading. These cations in CMF-DME did not support spreading in the absence of serum or the presence of serum albumin. Assaying cell spreading in a simple salt solution consisting of NaCl, KCl, Tris buffer, pH 7.4 plus dialyzed serum and a series of divalent cation supplements (Ca++, Mg++, Mn++, Co++, Zn++, Ni++), showed that only Mg++ and Mn++, and to a lesser extent, Ca++, supported cell spreading. In contrast to Mg++, however, Mn++ could support spreading in the absence of whole serum if serum albumin were present. Although Mn++ plus serum albumin supported more rapid spreading at lower cation concentrations than Mg++ plus serum, equal concentrations of Ca++ completely blocked the Mn++ effect. In contrast to the increasing cell spreading, which occurred in Mg++-containing medium with time, cell death occurred in Mn++-containing medium by 24 h. Consonant with studies from other laboratories, human foreskin fibroblasts spread in Mn++-containing salt solution in the absence of protein supplements. These experiments indicate for epidermal cell spreading that Mg++ is the important cation in tissue culture media, that under proper cation conditions epidermal cells do not need a specific spreading protein (i.e. a protein that has been demonstrated to support cell spreading), that Mn++ and Mg++-induced spreading seem to represent different mechanisms, that fibroblastic and epidermal cells have different cation requirements for in vitro spreading, and that the crucial role cations play in cell spreading remains to be elucidated.