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

We investigated whether alterations in the mechanisms involved in intracellular pH (pH(in)) and intracellular calcium ([Ca²⁺](in)) homeostasis are associated with the metastatic potential of poorly (A375P) and highly (C8161) metastatic human melanoma cells. We monitored pH(in) and [Ca²⁺](in) simultaneously, using the fluorescence of SNARF-1 and Fura-2, respectively. Our results indicated that steady-state pH(in) and [Ca²⁺](in) between these cell types were not significantly different. Treatment of cells with NH₄Cl resulted in larger pH(in) 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 ICa²⁺](in) regulatory mechanisms than C8161 cells, to recover from Ca²⁺ loads. Removal of extracellular Ca²⁺ ([Ca²⁺](in)) 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 (Martinez-Zaguilan et al., 1993). Collectively, these data suggest that distinct pH(in) and [Ca²⁺](in) regulatory mechanisms are present in poorly and highly metastatic human melanoma cells.