Distinct regulation of pHin and [Ca2+]in in human melanoma cells with different metastatic potential

We investigated whether alterations in the mechanisms involved in intracellular pH (pHⁱⁿ) and intracellular calcium (Ca²⁺]ⁱⁿ) homeostasis are associated with the metastatic potential of poorly (A375P) and highly (C8161) metastatic human melanoma cells. We monitored pHⁱⁿ and Ca²⁺]ⁱⁿ simultaneously, using the fluorescence of SNARF-1 and Fura-2, respectively. Our results indicated that steady-state pHⁱⁿ and Ca²⁺]ⁱⁿ between these cell types were not significantly different. Treatment of cells with NH₄Cl resulted in larger pHⁱⁿ increases in highly than in poorly metastatic cells, suggesting that C8161 cells have a lower H⁺ buffering capacity than A375P. NH₄Cl treatment also increased Ca²⁺]ⁱⁿ only in C8161 cells. To determine if the changes in Ca²⁺]ⁱⁿ triggered by NH₄Cl treatment were due to alterations in either H⁺- or Ca²⁺-buffering capacity, cells were treated with the Ca²⁺-ionophore 4Br-A23187, to alter Ca²⁺]ⁱⁿ. The magnitude of the ionophore-induced Ca²⁺]ⁱⁿ increase was slightly greater in C8161 cells than in A375P. Moreover, A375P cells recover from the ionophore-induced Ca²⁺]ⁱⁿ load, whereas C8161 cells did not, suggesting that A375P may exhibit distinct Ca²⁺]ⁱⁿ regulatory mechanisms than C8161 cells, to recover from Ca²⁺ loads. Removal of extracellular Ca²⁺ (Ca²⁺]^ex) decreased Ca²⁺]ⁱⁿ in both cell types at the same extent. Ionophore treatment in the absence of Ca²⁺]^ex transiently increased Ca²⁺]ⁱⁿ in C8161, but not in A375P cells. Endoplasmic reticulum (ER) Ca²⁺-ATPase inhibitors such as cyclopiazonic acid (CPA) and thapsigargin (TG) increased steady-state Ca²⁺]ⁱⁿ only in C8161 cells. Together, these data suggest that the contribution of intracellular Ca²⁺ stores for Ca²⁺]ⁱⁿ homeostasis is greater in highly than in poorly metastatic cells. Bafilomycin treatment, to inhibit V-type H⁺-ATPases, corroborated our previous results that V-H⁺-ATPases are functionally expressed at the plasma membranes of highly metastatic, but not in poorly metastatic cells in and Ca²⁺]ⁱⁿ regulatory mechanisms are present in poorly and highly metastatic human melanoma cells. J. Cell. Physiol. 176:196–205, 1998. © 1998 Wiley-Liss, Inc.