DISTINCT REGULATION OF PH(IN) AND [CA2-MELANOMA CELLS WITH DIFFERENT METASTATIC POTENTIAL(](IN) IN HUMAN)

We investigated whether alterations in the mechanisms involved in intr acellular pH (pH(in)) and intracellular calcium ([Ca2+](in)) homeostas is are associated with the metastatic potential of poorly (A375P) and highly (C8161) metastatic human melanoma cells. We monitored pH(in) an d [Ca2+](in) simultaneously, using the fluorescence of SNARF-1 and Fur a-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 h ighly than in poorly metastatic cells, suggesting that C8161 cells hav e a lower H+ buffering capacity than A375P. NH4Cl treatment also incre ased [Ca2+](in) only in C8161 cells. To determine if the changes in [C a2+](in) triggered by NH4Cl treatment were due to alterations in eithe r H+- or Ca2+-buffering capacity, cells were treated with the Ca2+-ion ophore 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 [Ca 2+](in) load, whereas C8161 cells did not, suggesting that A375P may e xhibit distinct [Ca2+](in) regulatory mechanisms than C8161 cells, to recover from Ca2+ loads. Removal of extracellular Ca2+ ([Ca2+](ex)) de creased [Ca2+](in) in both cell types at the same extent. Ionophore tr eatment in the absence of [Ca2+](ex) transiently increased [Ca2+](in) in C8161, but not in A375P cells. Endoplasmic reticulum (ER) Ca2+-ATPa se inhibitors such as cyclopiazonic acid (CPA) and thapsigargin (TC) i ncreased steady-state [Ca2+](in) only in C8161 cells. Together, these data suggest that the contribution of intracellular Ca2+ stores for [C a2+](in) homeostasis is greater in highly than in poorly metastatic ce lls. 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 metastati c cells (Martinez-Zaguilan et at., 1993). Collectively, these data sug gest that distinct pH(in) and [Ca2+](in) regulatory mechanisms are pre sent in poorly and highly metastatic human melanoma cells. (C) 1998 Wi ley-Liss, Inc.