We investigated whether alterations in the mechanisms involved in intracellular pH (pH
in) and intracellular calcium ([Ca
2+]
in) homeostasis are associated with the metastatic potential of poorly (A375P) and highly (C8161) metastatic human melanoma cells. We monitored pH
in and [Ca
2+]
in simultaneously, using the fluorescence of SNARF-1 and Fura-2, respectively. Our results indicated that steady-state pH
in and [Ca
2+]
in between these cell types were not significantly different. Treatment of cells with NH
4Cl 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. NH
4Cl treatment also increased [Ca
2+]
in only in C8161 cells. To determine if the changes in [Ca
2+]
in triggered by NH
4Cl treatment were due to alterations in either H
+- or Ca
2+-buffering capacity, cells were treated with the Ca
2+-ionophore 4Br-A23187, to alter [Ca
2+]
in. The magnitude of the ionophore-induced [Ca
2+]
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 exhibit distinct [Ca
2+]
in regulatory mechanisms than C8161 cells, to recover from Ca
2+ loads. Removal of extracellular Ca
2+ ([Ca
2+]
ex) decreased [Ca
2+]
in in both cell types at the same extent. Ionophore treatment in the absence of [Ca
2+]
ex transiently increased [Ca
2+]
in in C8161, but not in A375P cells. Endoplasmic reticulum (ER) Ca
2+-ATPase inhibitors such as cyclopiazonic acid (CPA) and thapsigargin (TG) increased steady-state [Ca
2+]
in only in C8161 cells. Together, these data suggest that the contribution of intracellular Ca
2+ stores for [Ca
2+]
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 [Ca
2+]
in regulatory mechanisms are present in poorly and highly metastatic human melanoma cells. J. Cell. Physiol. 176:196–205, 1998. © 1998 Wiley-Liss, Inc.
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